Downmodulating an immune response with multivalent antibodies to PD-1

ABSTRACT

The invention identifies PD-1 as a receptor for B7-4. B7-4 can inhibit immune cell activation upon binding to an inhibitory receptor on an immune cell. Accordingly, the invention provides agents for modulating PD-1, B7-4, and the interaction between B7-4 and PD-1 in order to modulate a costimulatory or an inhibitory signal in a immune cell resulting in modulation of the immune response.

RELATED APPLICATIONS

This claims priority to U.S. Ser. No. 60/150,390 filed on Aug. 23, 1999.This application also claims priority to U.S. Ser. No. 60/164,897, filedon Nov. 10, 1999. Both of these applications are incorporated herein intheir entirety by this reference.

GOVERNMENT FUNDING

Work described herein was supported under AI 39671, AI 44690, CA 84500and AI 41584 awarded by the National Institutes of Health. The U.S.government, therefore, may have certain rights in this invention.

BACKGROUND OF THE INVENTION

In order for T cells to respond to foreign proteins, two signals must beprovided by antigen-presenting cells (APCs) to resting T lymphocytes(Jenkins, M. and Schwartz, R. (1987) J. Exp. Med. 165:302-319; Mueller,D. L. et al. (1990) J. Immunol. 144:3701-3709). The first signal, whichconfers specificity to the immune response, is transduced via the T cellreceptor (TCR) following recognition of foreign antigenic peptidepresented in the context of the major histocompatibility complex (MHC).The second signal, termed costimulation, induces T cells to proliferateand become functional (Lenschow et al. (1996) Annu. Rev. Immunol.14:233). Costimulation is neither antigen-specific, nor MHC restrictedand is thought to be provided by one or more distinct cell surfacemolecules expressed by APCs (Jenkins, M. K. et al. (1988) J. Immunol.140:3324-3330; Linsley, P. S. et al. (1991) J. Exp. Med. 173:721-730;Gimmi, C. D., et al. 1991 Proc. Natl. Acad. Sci. USA 88:6575-6579;Young, J. W. et al. (1992) J. Clin. Invest. 90:229-237; Koulova, L. etal. (1991) J. Exp. Med. 173:759-762; Reiser, H. et al. (1992) Proc.Natl. Acad. Sci. USA 89:271-275; van-Seventer, G. A. et al. (1990) J.Immunol. 144:4579-4586; LaSalle, J. M. et al. (1991) J. Immunol.147:774-80; Dustin, M. I. et al. (1989) J. Exp. Med. 169:503; Armitage,R. J. et al. (1992) Nature 357:80-82; Liu, Y. et al. (1992) J. Exp. Med.175:437-445).

The CD80 (B7-1) and CD86 (B7-2) proteins, expressed on APCs, arecritical costimulatory molecules (Freeman et al. (1991) J. Exp. Med.174:625; Freeman et al. (1989) J. Immunol. 143:2714; Azuma et al. (1993)Nature 366:76; Freeman et al. (1993) Science 262:909). B7-2 appears toplay a predominant role during primary immune responses, while B7-1,which is upregulated later in the course of an immune response, may beimportant in prolonging primary T cell responses or costimulatingsecondary T cell responses (Bluestone (1995) Immunity 2:555).

One receptor to which B7-1 and B7-2 bind, CD28, is constitutivelyexpressed on resting T cells and increases in expression afteractivation. After signaling through the T cell receptor, ligation ofCD28 and transduction of a costimulatory signal induces T cells toproliferate and secrete IL-2 (Linsley, P. S. et al. (1991) J. Exp. Med.173:721-730; Gimmi, C. D. et al. (1991) Proc. Natl. Acad Sci. USA88:6575-6579; June, C. H. et al. (1990) Immunol. Today. 11:211-6;Harding, F. A. et al. (1992) Nature 356:607-609). A second receptor,termed CTLA4 (CD 152) is homologous to CD28 but is not expressed onresting T cells and appears following T cell activation (Brunet, J. F.et al. (1987) Nature 328:267-270). CTLA4 appears to be critical innegative regulation of T cell responses (Waterhouse et al. (1995)Science 270:985). Blockade of CTLA4 has been found to remove inhibitorysignals, while aggregation of CTLA4 has been found to provide inhibitorysignals that downregulate T cell responses (Allison and Krummel (1995)Science 270:932). The B7 molecules have a higher affinity for CTLA4 thanfor CD28 (Linsley, P. S. et al. (1991) J. Exp. Med. 174:561-569) andB7-1 and B7-2 have been found to bind to distinct regions of the CTLA4molecule and have different kinetics of binding to CTLA4 (Linsley et al.(1994) Immunity 1:793). A new molecule related to CD28 and CTLA4, ICOS,has been identified and seems to be important in IL-10 production(Hutloff et al. (1999) Nature 397:263; WO 98/38216), as has its ligand,which is a new B7 family member (Aicher A. et al. (2000) J. Immunol.164:4689-96; Mages H. W. et al. (2000) Eur. J. Immunol. 30:1040-7;Brodie D. et al. (2000) Curr. Biol. 10:333-6; Ling V. et al. (2000) J.Immunol. 164:1653-7; Yoshinaga S. K. et al. (1999) Nature 402:827-32).If T cells are only stimulated through the T cell receptor, withoutreceiving an additional costimulatory signal, they become nonresponsive,anergic, or die, resulting in downmodulation of the immune response.

The importance of the B7:CD28/CTLA4 costimulatory pathway has beendemonstrated in vitro and in several in vivo model systems. Blockade ofthis costimulatory pathway results in the development of antigenspecific tolerance in murine and human systems (Harding, F. A. et al.(1992) Nature 356:607-609; Lenschow, D. J. et al. (1992) Science257:789-792; Turka, L. A. et al. (1992) Proc. Natl. Acad. Sci. USA89:11102-11105; Gimmi, C. D. et al. (1993) Proc. Natl. Acad. Sci. USA90:6586-6590; Boussiotis, V. et al. (1993) J. Exp. Med. 178:1753-1763).Conversely, expression of B7 by B7 negative murine tumor cells inducesT-cell mediated specific immunity accompanied by tumor rejection andlong lasting protection to tumor challenge (Chen, L. et al. (1992) Cell71:1093-1102; Townsend, S. E. and Allison, J. P. (1993) Science259:368-370; Baskar, S. et al. (1993) Proc. Natl. Acad. Sci.90:5687-5690.). Therefore, manipulation of the costimulatory pathwaysoffers great potential to stimulate or suppress immune responses inhumans.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery thatPD-1 is a receptor for B7-4 molecules expressed on antigen presentingcells. PD-1 transmits a negative signal to immune cells, similar toCTLA4. B7-4 molecules are expressed on the surface of antigen presentingcells and provide a costimulatory signal to immune cells and cantransmit downmodulatory signals to immune cells, depending upon themolecule to which they bind. Thus, modulation of PD-1, B7-4, and/or theinteraction between B7-4 and PD-1 results in modulation of the immuneresponse.

Accordingly, in one aspect, the invention provides a method formodulating an immune response comprising contacting an immune cell withan agent that modulates signaling via PD-1 to thereby modulate theimmune response.

In one embodiment, the immune response is downregulated.

In another embodiment, signaling via PD-1 is stimulated using an agentselected from the group consisting of: an activating antibody thatrecognizes PD-1, a form of B7-4 that binds to an inhibitory receptor,and a small molecule that binds to PD-1.

In one embodiment, the immune cell is selected from the group consistingof: a T cell, a B cell, and a myeloid cell.

In one embodiment, anergy is induced in the immune cell.

In one embodiment, the method further comprising contacting the immunecell with an additional agent that downregulates an immune response.

In one embodiment, the immune response is upregulated.

In one embodiment, the signaling via PD-1 is inhibited using an agentselected from the group consisting of: a blocking antibody thatrecognizes PD-1, a non-activating form of B7-4, an antibody thatrecognizes B7-4, and a soluble form of PD-1.

In one embodiment, the step of contacting occurs in vivo. In anotherembodiment, the step of contacting occurs in vitro.

In another aspect, the invention pertains to a method for modulating theinteraction of B7-4 with an inhibitory receptor on an immune cellcomprising contacting an antigen presenting cell which expresses B7-4with an agent selected from the group consisting of: a form of B7-4, aform of PD-1, or an agent that modulates the interaction of B7-4 andPD-1 such that the interaction of B7-4 with an inhibitory receptor on animmune cell is modulated.

In one embodiment, the method further comprises contacting the immunecell or the antigen presenting cell with an additional agent thatmodulates an immune response.

In one embodiment, the step of contacting is performed in vitro. Inanother embodiment, the step of contacting is performed in vivo.

In one embodiment, the immune cell is selected from the group consistingof: a T cell, a B cell, and a myeloid cell.

In another aspect, the invention pertains to a method for inhibitingactivation in an immune cell via a non-apoptotic mechanism comprisingincreasing the activity or expression of PD-1 in a immune cell such thatimmune cell activation is inhibited.

In another aspect, the invention pertains to vaccine comprising anantigen and an agent that inhibits signaling via PD-1 in an immune cell.

In another aspect, the invention pertains to a composition comprising anantigen and an agent that promotes signaling via PD-1 in an immune cell.

In another aspect, the invention pertains to a method for treating asubject having a condition that would benefit from upregulation of animmune response comprising administering an agent that inhibitssignaling via PD-1 in a immune cell of the subject such that a conditionthat would benefit from upregulation of an immune response is treated.

In one embodiment, the agent comprises a soluble form of PD-1 or B7-4.

In one embodiment, the method further comprises administering a secondagent that upregulates an immune response to the subject.

In one embodiment, the condition is selected from the group consistingof: a tumor, a neurological disease or an immunosuppressive disease.

In another aspect, the invention pertains to a method for treating asubject having a condition that would benefit from downregulation of animmune response comprising administering an agent that stimulatessignaling via PD-1 in a immune cell of the subject such that a conditionthat would benefit from downregulation of an immune response is treated.

In one embodiment, said agent is selected from the group consisting of:an antibody that stimulates signaling via PD-1, a bispecific antibody,and soluble B7-4.

In one embodiment, the method further comprises administering a secondagent that downregulates an immune response to the subject.

In one embodiment, the condition is selected from the group consistingof: a transplant, an allergy, and an autoimmune disorder.

In another aspect, the invention pertains to a cell-based assay forscreening for compounds which modulate the activity of B7-4 or PD-1comprising contacting a cell expressing a B7-4 target molecule or PD-1target molecule with a test compound and determining the ability of thetest compound to modulate the activity of the B7-4 or PD-1 targetmolecule.

In yet another aspect, the invention pertains to a cell-free assay forscreening for compounds which modulate the binding of B7-4 or PD-1 to atarget molecule comprising contacting a B7-4 or PD-1 protein orbiologically active portion thereof with a test compound and determiningthe ability of the test compound to bind to the B7-4 or PD-1 protein orbiologically active portion thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence encoding a human secreted B7-4,B7-4S (SEQ ID NO: 1).

FIG. 2 depicts the nucleotide sequence encoding a human B7-4, B7-4M (SEQID NO: 3).

FIG. 3 depicts the amino acid sequence of human B7-4S (SEQ ID NO: 2) andillustrates the signal, IgV, IgC, and hydrophilic tail domains.

FIG. 4 depicts the amino acid sequence of human B7-4M (SEQ ID NO: 4) andillustrates the signal, IgV, IgC, and transmembrane and cytoplasmicdomains.

FIG. 5 depicts the nucleotide sequence of murine B7-4 (SEQ ID NO: 22).

FIG. 6 depicts the amino acid sequence of murine B7-4 (SEQ ID NO: 23).

FIG. 7 depicts an alignment of the human and murine B7-4 amino acidsequences (SEQ ID NO: 2 and 23 respectively).

FIG. 8 illustrates the results of FACS analysis of binding of CD28Ig,CTLA4-Ig, and control Ig by B7-4M-transfected COS cells.

FIG. 9 illustrates the results FACS analysis of binding of IgG andmurine ICOS-his fusion protein by B7-4M-transfected COS cells.

FIG. 10 illustrates the results FACS analysis of binding of IgM, BB1 and133 antibodies to B7-4M-transfected COS cells.

FIG. 11 illustrates that COS cells transfected with B7-4M (292) cancostimulate T cell proliferation.

FIG. 12 illustrates that COS cells transfected with a B7-4M (292) cancostimulate T cell proliferation.

FIG. 13 illustrates the binding of PD-1 to B7-4M transfected COS cells.

FIG. 14 illustrates the ability of added PD-1 and not Flt4 to competefor the binding of PD-1 to B7-4M transfected COS cells.

FIG. 15 illustrates the ability of PD-1 to bind to B7-4 transfected CHOcells, as determined by flow cytometry.

FIG. 16 illustrates the ability of PD-1 to bind to B7-4 transfected CHOcells, as determined by BIACORE analysis.

FIG. 17 illustrates the ability of B7-4M to transmit a negative signalto T cells.

FIG. 18 illustrates the inhibition of T cell proliferation and cytokineproduction in human T cell stimulated in the presence of B7-4.

FIG. 19 illustrates that T cell receptor/B7-4 activation in the presenceof CD28 costimulation results in inhibition of T cell proliferation.

FIG. 20 illustrates the binding of PD-1 to CHO cells expressing B7-4.

FIG. 21 illustrates the action of B7-4 in the inhibition of CD28signals.

FIG. 22 illustrates the inhibition of cytokine production by thePD-1:B7-4 pathway, as measured by cytokine ELISA.

FIG. 23 illustrates the inhibition of cytokine production by thePD-1:B7-4 pathway, as measured by cytokine mRNA levels.

FIG. 24 illustrates that the mechanism of action of the PD-1:B7-4pathway is cell-cycle arrest.

FIG. 25 illustrates the ability of antibodies to B7-4 to inhibit theinteraction between B7-4 and PD-1.

FIG. 26 illustrates the ability of antibodies to PD-1 to inhibit theinteraction between B7-4 and PD-1.

FIG. 27 illustrates the ability of soluble B7-4Fc to exacerbate diseasein a murine model of experimental autoimmune encephalomyelitis.

DETAILED DESCRIPTION OF THE INVENTION

In addition to the previously characterized B lymphocyte activationantigens, e.g., B7-1 and B7-2, there are other antigens on the surfaceof antigen presenting cells which modulate costimulation of immunecells. For example, B7-4 polypeptides have been isolated fromkeratinocyte and placental cDNA libraries. B7-4 has also been found toherein costimulate or inhibit T cells. The present invention identifiesPD-1 as a receptor for B7-4.

Immune cells have receptors that transmit activating signals. Forexample, T cells have T cell receptors and the CD3 complex, B cells haveB cell receptors, and myeloid cells have Fc receptors. In addition,immune cells bear receptors that transmit signals that providecostimulatory signals or receptors that transmit signals that inhibitreceptor-mediated signaling. For example, CD28 transmits a costimulatorysignal to T cells. After ligation of the T cell receptor, ligation ofCD28 results in a costimulatory signal characterized by, e.g.,upregulation of IL-2rα, IL-2rβ, and IL-2rγ receptor, increasedtranscription of IL-2 messenger RNA, increased expression of cytokinegenes (including IL-2, IFN-γ, GM-CSF, and TNF-a). Transmission of acostimulatory signal allows the cell to progress through the cell cycleand, thus, increases T cell proliferation (Greenfield et al. (1998)Crit. Rev. Immunol. 18:389). Binding of a receptor on a T cell whichtransmits a costimulatory signal to the cell (e.g., ligation of acostimulatory receptor that leads to cytokine secretion and/orproliferation of the T cell) by a B7 family molecule, such as B7-4,results in costimulation. Thus, inhibition of an interaction between aB7 family molecule, such as B7-4, and a receptor that transmits acostimulatory signal on a immune cells results in a downmodulation ofthe immune response and/or specific unresponsiveness, termed immune cellanergy. Inhibition of this interaction can be accomplished using, e.g.,anti-CD28 Fab fragments, antibodies to B7-1, B7-2 and/or B7-4, or byusing a soluble form of a receptor to which a B7 family member moleculecan bind as a competitive inhibitor (e.g., CTLA4Ig).

Inhibitory receptors that bind to costimulatory molecules have also beenidentified on immune cells. Activation of CTLA4, for example, transmitsa negative signal to a T cell. Engagement of CTLA4 inhibits IL-2production and can induce cell cycle arrest (Krummel and Allison (1996)J. Exp. Med. 183:2533). In addition, mice that lack CTLA4 developlymphoproliferative disease (Tivol et al. (1995) Immunity 3:541;Waterhouse et al. (1995) Science 270:985). The blockade of CTLA4 withantibodies may remove an inhibitory signal, whereas aggregation of CTLA4with antibody transmits an inhibitory signal. Therefore, depending uponthe receptor to which a costimulatory molecule binds (i.e., acostimulatory receptor such as CD28 or an inhibitory receptor such asCTLA4), certain B7 molecules can promote T cell costimulation orinhibition.

PD-1 is a member of the immunoglobulin family of molecules (Ishida etal. (1992) EMBO J. 11:3887; Shinohara et al. (1994) Genomics 23:704).PD-1 was previously identified using a subtraction cloning basedapproach designed to identify modulators of programmed cell death.(Ishida et al. (1992) EMBO J. 11:3887-95; Woronicz et al. (1995) Curr.Top. Microbiol. Immunol. 200:137). PD-1 is believed to play a role inlymphocyte survival, e.g., during clonal selection (Honjo (1992) Science258:591; Agata et al. (1996) Int. Immunology. 8:765; Nishimura et al.(1996) Int. Immunology 8:773). PD-1 was also implicated as a regulatorof B cell responses (Nishimura (1998) Int. Immunology 10:1563). UnlikeCTLA4, which is found only on T cells, PD-1 is also found on B cells andmyeloid cells.

The instant discovery that PD-1 binds to B7-4 places PD-1 in a family ofinhibitory receptors with CTLA4. While engagement of a costimulatoryreceptor results in a costimulatory signal in an immune cell, engagementof an inhibitory receptor, e.g., CTLA4 or PD-1 (for example bycrosslinking or by aggregation), leads to the transmission of aninhibitory signal in an immune cell, resulting in downmodulation ofimmune cell responses and/or in immune cell anergy. While transmissionof an inhibitory signal leads to downmodulation in immune cell responses(and a resulting downmodulation in the overall immune response), theprevention of an inhibitory signal (e.g., by using a non-activatingantibody against PD-1) in immune cells leads to upmodulation of immunecell responses (and a resulting upmodulation of an immune response).

The instant invention makes available agents useful for modulating theactivity and/or expression of PD-1; the interaction between PD-1 and itsnatural ligand(s), (e.g., B7-4); and agents for modulating the immuneresponse via modulation of the interaction between PD-1 and its naturalligand, e.g., B7-4. Exemplary modulatory agents for use in these methodsare described further as follows.

B7-4 and PD-1 Nucleic Acid and Polypeptide Molecules

In one embodiment, a modulatory agent useful for modulating the activityand/or expression of PD-1 is a B7-4 and/or PD-1 nucleic acid molecule,preferably a human B7-4 and/or PD-1 nucleic acid molecule.

In one embodiment, the isolated nucleic acid molecules of the presentinvention encode eukaryotic B7-4 or PD-1 polypeptides. The B7-4 familyof molecules share a number of conserved regions, including signaldomains, IgV domains and the IgC domains. IgV domains and the IgCdomains are art recognized Ig superfamily member domains. These domainscorrespond to structural units that have distinct folding patternscalled Ig folds. Ig folds are comprised of a sandwich of two β sheets,each consisting of antiparallel β strands of 5-10 amino acids with aconserved disulfide bond between the two sheets in most, but not all,domains. IgC domains of Ig, TCR, and MHC molecules share the same typesof sequence patterns and are called the C1-set within the Igsuperfamily. Other IgC domains fall within other sets. IgV domains alsoshare sequence patterns and are called V set domains. IgV domains arelonger than C-domains and form an additional pair of β strands.

Two forms of human B7-4 molecules have been identified. One form is anaturally occurring B7-4 soluble polypeptide, i.e., having a shorthydrophilic domain and no transmembrane domain, and is referred toherein as B7-4S (shown in SEQ ID NO:2). The second form is acell-associated polypeptide, i.e., having a transmembrane andcytoplasmic domain, referred to herein as B7-4M (shown in SEQ ID NO:4).

B7-4 proteins comprise a signal sequence, and an IgV domain and an IgCdomain. The signal sequence of SEQ ID NO:2 is shown from about aminoacid 1 to about amino acid 18. The signal sequence of SEQ ID NO:4 isshown from about amino acid 1 to about amino acid 18. The IgV domain ofSEQ ID NO:2 is shown from about amino acid 19 to about amino acid 134and the IgV domain of SEQ ID NO:4 is shown from about amino acid 19 toabout amino acid 134. The IgC domain of SEQ ID NO:2 is shown from aboutamino acid 135 to about amino acid 227 and the IgC domain of SEQ ID NO:4is shown from about amino acid 135 to about amino acid 227. Thehydrophilic tail of the B7-4 exemplified in SEQ ID NO:2 comprises ahydrophilic tail shown from about amino acid 228 to about amino acid245. The B7-4 polypeptide exemplified in SEQ ID NO:4 comprises atransmembrane domain shown from about amino acids 239 to about aminoacid 259 of SEQ ID NO:4 and a cytoplasmic domain shown from about aminoacid 260 to about amino acid 290 of SEQ ID NO:4.

Murine B7-4 molecules were also identified. The murine cDNA sequence ispresented in FIG. 5 and the murine B7-4 amino acid sequence is presentedin FIG. 6. The present invention also pertains to these murine B7-4molecules.

PD-1 has been identified herein as a receptor which binds to B7-4. PD-1molecules are members of the immunoglobulin gene superfamily. PD-1(Ishida et al. (1992) EMBO J. 11:3887; Shinohara et al. (1994) Genomics23:704; U.S. Pat. No. 5,698,520) has an extracellular region containingimmunoglobulin superfamily domain, a transmembrane domain, and anintracellular region including an immunoreceptor tyrosine-basedinhibitory motif (ITIM). These features also define a larger family ofmolecules, called the immunoinhibitory receptors, which also includesgp49B, PIR-B, and the killer inhibitory receptors (KIRs) (Vivier andDaeron (1997) Immunol. Today 18:286). It is often assumed that thetyrosyl phosphorylated ITIM motif of these receptors interacts withSH2-domain containing phosphatases, which leads to inhibitory signals. Asubset of these immunoinhibitory receptors bind to MHC molecules, forexample the KIRs, and CTLA4 bind to B7-1 and B7-2. It has been proposedthat there is a phylogenetic relationship between the MHC and B7 genes(Henry et al. (1999) Immunol. Today 20(6):285-8).

The nucleotide sequence of PD-1 is shown in SEQ ID NO:10 and 11 and theamino acid sequence of PD-1 is shown in SEQ ID NO:12 (see also Ishida etal. (1992) EMBO J. 11:3887; Shinohara et al. (1994) Genomics 23:704;U.S. Pat. No. 5,698,520). PD-1 was previously identified using asubtraction cloning based approach to select for proteins involved inapoptotic cell death. PD-1 is identified herein as a member of theCD28/CTLA-4 family of molecules based on its ability to bind to B7-4.Like CTLA4, PD-1 is rapidly induced on the surface of T-cells inresponse to anti-CD3 (Agata et al. (1996) Int. Immunol. 8:765). Incontrast to CTLA4, however, PD-1 is also induced on the surface ofB-cells (in response to anti-IgM). PD-1 is also expressed on a subset ofthymocytes and myeloid cells (Agata et al. (1996) supra; Nishimura etal. (1996) Int. Immunol. 8:773). The instant invention identifies B7-4as a ligand of PD-1.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Definitions

As used herein, the term “immune cell” includes cells that are ofhematopoietic origin and that play a role in the immune response. Immunecells include lymphocytes, such as B cells and T cells; natural killercells; myeloid cells, such as monocytes, macrophages, eosinophils, mastcells, basophils, and granulocytes.

As used herein, the term “T cell” includes CD4+ T cells and CD8+ Tcells. The term T cell also includes both T helper 1 type T cells and Thelper 2 type T cells. The term “antigen presenting cell” includesprofessional antigen presenting cells (e.g., B lymphocytes, monocytes,dendritic cells, Langerhans cells) as well as other antigen presentingcells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes).

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune responses that are influenced bymodulation of T cell costimulation. Exemplary immune responses include Tcell responses, e.g., cytokine production, and cellular cytotoxicity. Inaddition, the term immune response includes immune responses that areindirectly effected by T cell activation, e.g., antibody production(humoral responses) and activation of cytokine responsive cells, e.g.,macrophages.

As used herein, the term “costimulatory receptor” includes receptorswhich transmit a costimulatory signal to a immune cell, e.g., CD28. Asused herein, the term “inhibitory receptors” includes receptors whichtransmit a negative signal to an immune cell (e.g., CTLA4 or PD-1). Aninhibitory signal as transduced by an inhibitory receptor can occur evenif a costimulatory receptor (such as CD28) in not present on the immunecell and, thus, is not simply a function of competition betweeninhibitory receptors and costimulatory receptors for binding ofcostimulatory molecules (Fallarino et al. (1998) J. Exp. Med. 188:205).Transmission of an inhibitory signal to an immune cell can result inunresponsiveness or anergy or programmed cell death in the immune cell.Preferably transmission of an inhibitory signal operates through amechanism that does not involve apoptosis. As used herein the term“apoptosis” includes programmed cell death which can be characterizedusing techniques which are known in the art. Apoptotic cell death can becharacterized, e.g., by cell shrinkage, membrane blebbing and chromatincondensation culminating in cell fragmentation. Cells undergoingapoptosis also display a characteristic pattern of internucleosomal DNAcleavage.

Depending upon the form of the B7-4 molecule that binds to a receptor,either a signal can be transmitted (e.g., by a multivalent form of aB7-4 molecule that results in crosslinking of receptor) or a signal canbe inhibited (e.g., by a soluble, monovalent form of a B7-4 molecule),e.g., by competing with activating forms of B7-4 molecules for bindingto the receptor. However, there are instances in which a solublemolecule can be stimulatory. The effects of the various modulatoryagents can be easily demonstrated using routine screening assays asdescribed herein.

As used herein, the term “costimulate” with reference to activatedimmune cells includes the ability of a costimulatory molecule to providea second, non-activating receptor mediated signal (a “costimulatorysignal”) that induces proliferation or effector function. For example, acostimulatory signal can result in cytokine secretion, e.g., in a T cellthat has received a T cell-receptor-mediated signal. Immune cells thathave received a cell-receptor mediated signal, e.g., via an activatingreceptor are referred to herein as “activated immune cells.”

As used herein, the term “activating receptor” includes immune cellreceptors that bind antigen, complexed antigen (e.g., in the context ofMHC molecules), or bind to antibodies. Such activating receptors includeT cell receptors (TCR), B cell receptors (BCR), cytokine receptors, LPSreceptors, complement receptors, and Fc receptors.

For example, T cell receptors are present on T cells and are associatedwith CD3 molecules. T cell receptors are stimulated by antigen in thecontext of MHC molecules (as well as by polyclonal T cell activatingreagents). T cell activation via the TCR results in numerous changes,e.g., protein phosphorylation, membrane lipid changes, ion fluxes,cyclic nucleotide alterations, RNA transcription changes, proteinsynthesis changes, and cell volume changes.

B cell receptors are present on B cells. B cell antigen receptors are acomplex between membrane Ig (mIg) and other transmembrane polypeptides(e.g., Igα and Igβ). The signal transduction function of mIg istriggered by crosslinking of receptor molecules by oligomeric ormultimeric antigens. B cells can also be activated byanti-immunoglobulin antibodies. Upon BCR activation, numerous changesoccur in B cells, including tyrosine phosphorylation.

Fc receptors are found on many cells which participate in immuneresponses. Fc receptors (FcRs) are cell surface receptors for the Fcportion of immunoglobulin molecules (Igs). Among the human FcRs thathave been identified so far are those which recognize IgG (designatedFcγ R), IgE (Fcε R1), IgA (Fcα), and polymerized IgM/A (Fcμα R). FcRsare found in the following cell types: Fcε R I (mast cells), Fcε R.II(many leukocytes), Fcα R (neutrophils), and Fcμα R (glandularepithelium, hepatocytes) (Hogg, N. (1988) Immunol. Today 9:185-86). Thewidely studied FcγRs are central in cellular immune defenses, and areresponsible for stimulating the release of mediators of inflammation andhydrolytic enzymes involved in the pathogenesis of autoimmune disease(Unkeless, J. C. et al. (1988) Annu. Rev. Immunol. 6:251-81). The FcγRsprovide a crucial link between effector cells and the lymphocytes thatsecrete Ig, since the macrophage/monocyte, polymorphonuclear leukocyte,and natural killer (NK) cell FcγRs confer an element of specificrecognition mediated by IgG. Human leukocytes have at least threedifferent receptors for IgG: h Fcγ RI (found on monocytes/macrophages),hFcγ RII (on monocytes, neutrophils, eosinophils, platelets, possibly Bcells, and the K562 cell line), and Fcγ III (on NK cells, neutrophils,eosinophils, and macrophages).

With respect to T cells, transmission of a costimulatory signal to a Tcell involves a signaling pathway that is not inhibited by cyclosporineA. In addition, a costimulatory signal can induce cytokine secretion(e.g., IL-2 and/or IL-10) in a T cell and/or can prevent the inductionof unresponsiveness to antigen, the induction of anergy, or theinduction of cell death in the T cell.

As used herein, the term “inhibitory signal” refers to a signaltransmitted via an inhibitory receptor (e.g., CTLA4 or PD-1) for amolecule on a immune cell. Such a signal antagonizes a signal via anactivating receptor (e.g., via a TCR, CD3, BCR, or Fc molecule) and canresult in, e.g., inhibition of second messenger generation; aninhibition of proliferation; an inhibition of effector function in theimmune cell, e.g., reduced phagocytosis, reduced antibody production,reduced cellular cytotoxicity, the failure of the immune cell to producemediators, (such as cytokines (e.g., IL-2) and/or mediators of allergicresponses); or the development of anergy.

As used herein, the term “unresponsiveness” includes refractivity ofimmune cells to stimulation, e.g., stimulation via an activatingreceptor or a cytokine. Unresponsiveness can occur, e.g., because ofexposure to immunosuppressants or exposure to high doses of antigen. Asused herein, the term “anergy” or “tolerance” includes refractivity toactivating receptor-mediated stimulation. Such refractivity is generallyantigen-specific and persists after exposure to the tolerizing antigenhas ceased. For example, anergy in T cells (as opposed tounresponsiveness) is characterized by lack of cytokine production, e.g.,IL-2. T cell anergy occurs when T cells are exposed to antigen andreceive a first signal (a T cell receptor or CD-3 mediated signal) inthe absence of a second signal (a costimulatory signal). Under theseconditions, reexposure of the cells to the same antigen (even ifreexposure occurs in the presence of a costimulatory molecule) resultsin failure to produce cytokines and, thus, failure to proliferate.Anergic T cells can, however, mount responses to unrelated antigens andcan proliferate if cultured with cytokines (e.g., IL-2). For example, Tcell anergy can also be observed by the lack of IL-2 production by Tlymphocytes as measured by ELISA or by a proliferation assay using anindicator cell line. Alternatively, a reporter gene construct can beused. For example, anergic T cells fail to initiate IL-2 genetranscription induced by a heterologous promoter under the control ofthe 5′ IL-2 gene enhancer or by a multimer of the AP1 sequence that canbe found within the enhancer (Kang et al. (1992) Science 257:1134).

The B7-4 protein and nucleic acid molecules comprise a family ofmolecules having certain conserved structural and functional features.Similarly, the PD-1 protein and nucleic acid molecules are members of afamily of molecules having conserved structural and functional features.The term “family” when referring to the protein and nucleic acidmolecules is intended to mean two or more proteins or nucleic acidmolecules having a common structural domain or motif and havingsufficient amino acid or nucleotide sequence homology as defined herein.Such family members can be naturally or non-naturally occurring and canbe from either the same or different species. For example, a family cancontain a first protein of human origin, as well as other, distinctproteins of human origin or alternatively, can contain homologues ofnon-human origin. Members of a family may also have common functionalcharacteristics. The B7-4 molecules described herein are members of theB7 family of molecules. The term “B7 family” or “B7 molecules” as usedherein includes costimulatory molecules that share sequence homologywith B7 polypeptides, e.g., with B7-1, B7-2, B7-3 (recognized by theantibody BB-1), B7h (Swallow et al. (1999) Immunity 11:423), and/orB7-4. For example, human B7-1 and B7-2 share approximately 26% aminoacid sequence identity when compared using the BLAST program at NCBIwith the default parameters (Blosum62 matrix with gap penalties set atexistence 11 and extension 1 (See the NCBI website)).

Preferred B7 polypeptides are capable of providing costimulatory orinhibitory signals to immune cells to thereby promote or inhibit immunecell responses. For example, when bound to a costimulatory receptor,B7-4 can induce costimulation of immune cells or can inhibit immune cellcostimulation, e.g., when present in soluble form. When bound to aninhibitory receptor, B7-4 molecules can transmit an inhibitory signal toan immune cell. Preferred B7 family members include B7-1, B7-2, B7-3(recognized by the antibody BB-1), B7h, and B7-4 and soluble fragmentsor derivatives thereof. In one embodiment, B7 family members bind to oneor more receptors on an immune cell, e.g., CTLA4, CD28, ICOS, PD-1and/or other receptors, and, depending on the receptor, have the abilityto transmit an inhibitory signal or a costimulatory signal to an immunecell, preferably a T cell.

Preferred PD-1 molecules are capable of transmitting an inhibitorysignal to an immune cell to thereby inhibit immune cell effectorfunction or are capable of promoting costimulation (e.g., by competitiveinhibition) of immune cells, e.g., when present in soluble, monomericform. Preferred PD-1 family members bind to one or more receptors, e.g.,B7-1, B7-2, B7-4, and/or other molecules on antigen presenting cells,and share sequence identity with PD-1.

In addition, in one embodiment, proteins that are members of a proteinfamily are bound by antibodies generated against one or more otherfamily member proteins. For example, the anti-BB1 antibody recognizesB7-4 molecules.

As used herein, the term “activity” with respect to a B7-4 or PD-1polypeptide includes activities which are inherent in the structure of aB7-4 or PD-1 protein. With regard to B7-4, the term “activity” includesthe ability to modulate immune cell costimulation, e.g. by modulating acostimulatory signal in an activated immune cell, or to modulateinhibition by modulating an inhibitory signal in an immune cell, e.g.,by engaging a natural receptor on a immune cell. When an activating formof the B7-4 molecule binds to a costimulatory receptor, a costimulatorysignal is generated in the immune cell. When an activating form of theB7-4 molecule binds to an inhibitory receptor, an inhibitory signal isgenerated in the immune cell.

Modulation of a costimulatory signal results in modulation of effectorfunction of an immune cell. Thus, the term “B7-4 activity” includes theability of a B7-4 polypeptide to bind its natural receptor(s), theability to modulate immune cell costimulatory or inhibitory signals, andthe ability to modulate the immune response.

With respect to PD-1, the term “activity” includes the ability of a PD-1polypeptide to modulate an inhibitory signal in an activated immunecell, e.g., by engaging a natural ligand on an antigen presenting cell.PD-1 transmits an inhibitory signal to an immune cell in a mannersimilar to CTLA4. Modulation of an inhibitory signal in an immune cellresults in modulation of proliferation of and/or cytokine secretion byan immune cell. PD-1 can also modulate a costimulatory signal bycompeting with a costimulatory receptor for binding of a B7 molecule.Thus, the term “PD-1 activity” includes the ability of a PD-1polypeptide to bind its natural ligand(s), the ability to modulateimmune cell costimulatory or inhibitory signals, and the ability tomodulate the immune response.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

As used herein, an “antisense” nucleic acid molecule comprises anucleotide sequence which is complementary to a “sense” nucleic acidencoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA molecule, complementary to an mRNA sequence orcomplementary to the coding strand of a gene. Accordingly, an antisensenucleic acid molecule can hydrogen bond to a sense nucleic acidmolecule.

As used herein, the term “coding region” refers to regions of anucleotide sequence comprising codons which are translated into aminoacid residues, whereas the term “noncoding region” refers to regions ofa nucleotide sequence that are not translated into amino acids (e.g., 5′and 3′ untranslated regions).

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid molecule to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”or simply “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

As used herein, the term “host cell” is intended to refer to a cell intowhich a nucleic acid molecule of the invention, such as a recombinantexpression vector of the invention, has been introduced. The terms “hostcell” and “recombinant host cell” are used interchangeably herein. Itshould be understood that such terms refer not only to the particularsubject cell but to the progeny or potential progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term as used herein.

As used herein, a “transgenic animal” refers to a non-human animal,preferably a mammal, more preferably a mouse, in which one or more ofthe cells of the animal includes a “transgene”. The term “transgene”refers to exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, for example directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal.

As used herein, a “homologous recombinant animal” refers to a type oftransgenic non-human animal, preferably a mammal, more preferably amouse, in which an endogenous gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

As used herein, an “isolated protein” refers to a protein that issubstantially free of other proteins, cellular material and culturemedium when isolated from cells or produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized. An “isolated” or “purified” protein or biologically activeportion thereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theB7-4 or PD-1 protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of B7-4or PD-1 protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of B7-4 or PD-1 protein havingless than about 30% (by dry weight) of non-B7-4 or PD-1 protein (alsoreferred to herein as a “contaminating protein”), more preferably lessthan about 20% of non-B7-4 or PD-1 protein, still more preferably lessthan about 10% of non-B7-4 or PD-1 protein, and most preferably lessthan about 5% non-B7-4 or PD-1 protein. When the B7-4 or PD-1 protein orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of B7-4 or PD-1 protein in which theprotein is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of B7-4 or PD-1 protein having less than about 30%(by dry weight) of chemical precursors or non-B7-4 or PD-1 chemicals,more preferably less than about 20% chemical precursors or non-B7-4 orPD-1 chemicals, still more preferably less than about 10% chemicalprecursors or non-B7-4 or PD-1 chemicals, and most preferably less thanabout 5% chemical precursors or non-B7-4 or PD-1 chemicals.

The term “antibody” as used herein also includes an “antigen-bindingportion” of an antibody (or simply “antibody portion”). The term“antigen-binding portion”, as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., B7-4). It has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546 ), which consists of a VHdomain; and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778).Such single chain antibodies are also intended to be encompassed withinthe term “antigen-binding portion” of an antibody. Any VH and VLsequences of specific scFv can be linked to human immunoglobulinconstant region cDNA or genomic sequences, in order to generateexpression vectors encoding complete IgG molecules or other isotypes. VHand V1 can also be used in the generation of Fab, Fv or other fragmentsof immunoglobulins using either protein chemistry or recombinant DNAtechnology. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g. Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, orsyngeneic; or modified forms thereof, e.g. humanized, chimeric, etc.Preferably, antibodies of the invention bind specifically orsubstantially specifically to B7-4 molecules. The terms “monoclonalantibodies” and “monoclonal antibody composition”, as used herein, referto a population of antibody molecules that contain only one species ofan antigen binding site capable of immunoreacting with a particularepitope of an antigen, whereas the term “polyclonal antibodies” and“polyclonal antibody composition” refer to a population of antibodymolecules that contain multiple species of antigen binding sites capableof interacting with a particular antigen. A monoclonal antibodycomposition, typically displays a single binding affinity for aparticular antigen with which it immunoreacts.

The term “humanized antibody”, as used herein, is intended to includeantibodies made by a non-human cell having variable and constant regionswhich have been altered to more closely resemble antibodies that wouldbe made by a human cell. For example, by altering the non-human antibodyamino acid sequence to incorporate amino acids found in human germlineimmunoglobulin sequences. The humanized antibodies of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs. The term “humanized antibody”, as used herein, also includesantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds B7-4 is substantially free of antibodies that specifically bindantigens other than B7-4). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

There is a known and definite correspondence between the amino acidsequence of a particular protein and the nucleotide sequences that cancode for the protein, as defined by the genetic code (shown below).Likewise, there is a known and definite correspondence between thenucleotide sequence of a particular nucleic acid molecule and the aminoacid sequence encoded by that nucleic acid molecule, as defined by thegenetic code.

GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R) AGA,ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT Aspartic acid (Asp,D) GAC, GAT Cysteine (Cys, C) TGC, TGT Glutamic acid (Glu, E) GAA, GAGGlutamine (Gln, Q) CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGTHistidine (His, H) CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATT Leucine(Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAGMethionine (Met, M) ATG Phenylalanine (Phe, P) TTC, TTT Proline (Pro, P)CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCTThreonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine(Tyr, Y) TAC, TAT Valine (Val, V) GTA, GTC, GTG, GTT Termination signal(end) TAA, TAG, TGA

An important and well known feature of the genetic code is itsredundancy, whereby, for most of the amino acids used to make proteins,more than one coding nucleotide triplet may be employed (illustratedabove). Therefore, a number of different nucleotide sequences may codefor a given amino acid sequence. Such nucleotide sequences areconsidered functionally equivalent since they result in the productionof the same amino acid sequence in all organisms (although certainorganisms may translate some sequences more efficiently than they doothers). Moreover, occasionally, a methylated variant of a purine orpyrimidine may be found in a given nucleotide sequence. Suchmethylations do not affect the coding relationship between thetrinucleotide codon and the corresponding amino acid.

In view of the foregoing, the nucleotide sequence of a DNA or RNAmolecule coding for a B7-4 or PD-1 polypeptide of the invention (or anyportion thereof) can be used to derive the B7-4 or PD-1 amino acidsequence, using the genetic code to translate the DNA or RNA moleculeinto an amino acid sequence. Likewise, for any B7-4 or PD-1-amino acidsequence, corresponding nucleotide sequences that can encode B7-4 orPD-1 protein can be deduced from the genetic code (which, because of itsredundancy, will produce multiple nucleic acid sequences for any givenamino acid sequence). Thus, description and/or disclosure herein of aB7-4 or PD-1 nucleotide sequence should be considered to also includedescription and/or disclosure of the amino acid sequence encoded by thenucleotide sequence. Similarly, description and/or disclosure of a B7-4or PD-1 amino acid sequence herein should be considered to also includedescription and/or disclosure of all possible nucleotide sequences thatcan encode the amino acid sequence.

II. Isolated Nucleic Acid Molecules

In one embodiment, modulating agents for use in the claimed methodscomprise isolated nucleic acid molecules that encode B7-4 or PD-1proteins or biologically active portions thereof. Nucleic acid fragmentssufficient for use as hybridization probes to identify B7-4 orPD-1-encoding nucleic acids (e.g., B7-4 or PD-1 mRNA) and fragments foruse as PCR primers for the amplification or mutation of B7-4 or PD-1nucleic acid molecules are also provided. As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. For example, with regards to genomic DNA, theterm “isolated” includes nucleic acid molecules which are separated fromthe chromosome with which the genomic DNA is naturally associated.Preferably, an “isolated” nucleic acid molecule is free of sequenceswhich naturally flank the nucleic acid molecule (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid molecule is derived. For example,in various embodiments, the isolated B7-4 or PD-1 nucleic acid moleculecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1kb of nucleotide sequences which naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. An “isolated” B7-4 or PD-1 nucleic acid molecule may,however, be linked to other nucleotide sequences that do not normallyflank the B7-4 or PD-1 sequences in genomic DNA (e.g., the B7-4 or PD-1nucleotide sequences may be linked to vector sequences). In certainpreferred embodiments, an “isolated” nucleic acid molecule, such as acDNA molecule, also may be free of other cellular material. However, itis not necessary for the B7-4 or PD-1 nucleic acid molecule to be freeof other cellular material to be considered “isolated” (e.g., a B7-4 orPD-1 DNA molecule separated from other mammalian DNA and inserted into abacterial cell would still be considered to be “isolated”).

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, 3, 10, or 11 ora portion thereof, can be isolated using standard molecular biologytechniques and the sequence information provided herein. For example,using all or portion of the nucleic acid sequence of SEQ ID NO:1, 3, 10,or I 1, as a hybridization probe, B7-4 or PD-1 nucleic acid moleculescan be isolated using standard hybridization and cloning techniques(e.g., as described in Sambrook, J. et al. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1, 3, 10, or 11 can be isolated by the polymerase chain reaction(PCR) using synthetic oligonucleotide primers designed based upon thesequence of SEQ ID NO:1, 3, 10, or 11, respectively.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to B7-4 or PD-1 nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:1, 3, 10,or 11.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:1, 3, 10, or 11, or a portionof any of these nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:1, 3, 10, or11, is one which is sufficiently complementary to the nucleotidesequence shown in SEQ ID NO:1, 3, 10, or 11, respectively, such that itcan hybridize to the nucleotide sequence shown in SEQ ID NO:1, 3, 10, or11, respectively, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% ormore homologous to the nucleotide sequence (e.g., to the entire lengthof the nucleotide sequence) shown in SEQ ID NO:1, 3, 10, or 11, or aportion of any of these nucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, 3, 10, or 11, forexample a fragment which can be used as a probe or primer or a fragmentencoding a biologically active portion of a B7-4 or PD-1 protein. Thenucleotide sequence determined from the cloning of the B7-4 or PD-1genes allows for the generation of probes and primers designed for usein identifying and/or cloning other B7-4 or PD-1 family members, as wellas B7-4 or PD-1 family homologues from other species. The probe/primertypically comprises a substantially purified oligonucleotide. Theoligonucleotide typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12 or 15,preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55,60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ IDNO:1, 3, 10, or 11, or of a naturally occurring allelic variant ormutant of SEQ ID NO:1, 3, 10, or 11. In an exemplary embodiment, anucleic acid molecule of the present invention comprises a nucleotidesequence which is at least 350, 400, 450, 500, 550, 600, 650, 700, 750,or 800 nucleotides in length and hybridizes under stringenthybridization conditions to a nucleic acid molecule of SEQ ID NO:1, 3,10, or 11.

In another embodiment, a second nucleic acid molecule comprises at leastabout 500, 600, 700, 800, 900, or 1000 contiguous nucleotides of SEQ IDNO:1, 3, 10, or 11.

In one embodiment, a nucleic acid molecule of the invention, e.g., foruse as a probe, does not include the portion of SEQ ID NO:1 from aboutnucleotides 815 to about 850 of SEQ ID NO:1 or about nucleotides 320 to856 of SEQ ID NO:1. In another embodiment, a nucleic acid molecule ofthe invention does not include the portion of SEQ ID NO:3 from aboutnucleotides 314 to about 734, or from about nucleotides 835 to about860, or from about nucleotides 1085 to about 1104 or from aboutnucleotides 1286 to about 1536 of SEQ ID NO:3.

In one embodiment, a nucleic acid molecule of the invention comprises atleast about 500 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:3. Ina preferred embodiment, a nucleic acid molecule of the inventioncomprises at least about 600, at least about 700, at least about 800, atleast about 900 or at least about 950 contiguous nucleotides of SEQ IDNO:1 or about 1000 contiguous nucleotides of SEQ ID NO:3. In anotherembodiment, a nucleic acid molecule of the invention comprises at leastabout 1500 or 1550 nucleotides of SEQ ID NO:3.

Preferably, an isolated nucleic acid molecule of the invention comprisesat least a portion of the coding region of SEQ ID NO:1 (shown innucleotides 59-793) or SEQ ID NO:3 (shown in nucleotides 53-922). Inanother embodiment, a B7-4 nucleic acid molecule comprises from aboutnucleotide 1 to about nucleotide 319 of SEQ ID NO:1. In anotherembodiment, a B7-4 nucleic acid molecule comprises from about nucleotide855 to about nucleotide 968 of SEQ ID NO:1. In another embodiment, aB7-4 nucleic acid molecule comprises from about nucleotide 1 to aboutnucleotide 314 of SEQ ID NO:3. In another embodiment, a B7-4 nucleicacid molecule comprises from about nucleotide 955 to about nucleotide1285 of SEQ ID NO:3. In another embodiment, a B7-4 nucleic acid moleculecomprises from about nucleotide 1535 to about nucleotide 1552 of SEQ IDNO:3.

In other embodiments, a nucleic acid molecule of the invention has atleast 70% identity, more preferably 80% identity, and even morepreferably 90% identity with a nucleic acid molecule comprising: atleast about 500, at least about 600, at least about 700, at least about800, at least about 900 or at least about 1000 contiguous nucleotides ofSEQ ID NO:1 or SEQ ID NO:3.

Probes based on the B7-4 or PD-1 nucleotide sequences can be used todetect transcripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissues which misexpress a B7-4 or PD-1 protein, such as by measuring alevel of a B7-4 or PD-1-encoding nucleic acid in a sample of cells froma subject e.g., detecting B7-4 or PD-1 mRNA levels or determiningwhether a genomic B7-4 or PD-1 gene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of aB7-4 or PD-1 protein” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO:1, 3, 10, or 11 which encodes apolypeptide having a B7-4 or PD-1 biological activity (the biologicalactivities of the B7-4 or PD-1 proteins are described herein),expressing the encoded portion of the B7-4 or PD-1 protein (e.g., byrecombinant expression in vitro) and assessing the activity of theencoded portion of the B7-4 or PD-1 protein.

Nucleic acid molecules that differ from SEQ ID NO:1, 3, 10, or 11 due todegeneracy of the genetic code, and thus encode the same B7-4 or PD-1protein as that encoded by SEQ ID NO:1, 3, 10, or 11, are encompassed bythe invention. Accordingly, in another embodiment, an isolated nucleicacid molecule of the invention has a nucleotide sequence encoding aprotein having an amino acid sequence shown in SEQ ID NO:2, 4, or 12. Inanother embodiment, an isolated nucleic acid molecule of the inventionhas a nucleotide sequence encoding a B7-4 or PD-1 protein.

In addition to the B7-4 or PD-1 nucleotide sequences shown in SEQ IDNO:1, 3, 10, or 11 it should be appreciated by those skilled in the artthat DNA sequence polymorphisms that lead to changes in the amino acidsequences of the B7-4or PD-1 proteins may exist within a population(e.g., the human population). Such genetic polymorphism in the B7-4 orPD-1 genes may exist among individuals within a population due tonatural allelic variation. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules which include an openreading frame encoding a B7-4 or PD-1 protein, preferably a mammalianB7-4 or PD-1 protein, and can further include non-coding regulatorysequences, and introns. Such natural allelic variations include bothfunctional and non-functional B7-4 or PD-1 proteins and can typicallyresult in 1-5% variance in the nucleotide sequence of a B7-4 or PD-1gene. Such nucleotide variations and resulting amino acid polymorphismsin B7-4 or PD-1 genes that are the result of natural allelic variationand that do not alter the functional activity of a B7-4 or PD-1 proteinare intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding other B7-4 or PD-1 familymembers and, thus, which have a nucleotide sequence which differs fromthe B7-4 or PD-1 family sequences of SEQ ID NO:1, 3, 10, or 11 areintended to be within the scope of the invention. For example, anotherB7-4 or PD-1 cDNA can be identified based on the nucleotide sequence ofhuman B7-4 or PD-1. Moreover, nucleic acid molecules encoding B7-4 orPD-1 proteins from different species, and thus which have a nucleotidesequence which differs from the B7-4 or PD-1 sequences of SEQ ID NO:1,3, 10, or II are intended to be within the scope of the invention. Forexample, a mouse B7-4 or PD-1 cDNA can be identified based on thenucleotide sequence of a human B7-4 or PD-1 molecule.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the B7-4 or PD-1 cDNAs of the invention can be isolatedbased on their homology to the B7-4 or PD-1 nucleic acids disclosedherein using the cDNAs disclosed herein, or a portion thereof, as ahybridization probe according to standard hybridization techniques. Forexample, a B7-4 or PD-1 DNA can be isolated from a human genomic DNAlibrary using all or portion of SEQ ID NO:1, 3, 10, or 11 as ahybridization probe and standard hybridization techniques (e.g., asdescribed in Sambrook, J., et al. Molecular Cloning: A LaboratoryManual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989). Moreover, a nucleic acid molecule encompassing all or aportion of a B7-4 or PD-1 gene can be isolated by the polymerase chainreaction using oligonucleotide primers designed based upon the sequenceof SEQ ID NO:1, 3, 10, or 11. For example, mRNA can be isolated fromcells (e.g., by the guanidinium-thiocyanate extraction procedure ofChirgwin et al. (1979) Biochemistry 18:5294-5299) and cDNA can beprepared using reverse transcriptase (e.g., Moloney MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for PCR amplification can bedesigned based upon the nucleotide sequence shown in SEQ ID NO:1, 3, 10,or 11. A nucleic acid molecule of the invention can be amplified usingcDNA or, alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to a B7-4 or PD-1 nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In another embodiment, an isolated nucleic acid molecule of theinvention is at least 15, 20, 25, 30 or more nucleotides in length andhybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, 3, 10, or 11. Inother embodiment, the nucleic acid molecule is at least 30, 50, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 nucleotides inlength. As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 30%, 40%, 50%, or 60% homologous toeach other typically remain hybridized to each other. Preferably, theconditions are such that sequences at least about 70%, more preferablyat least about 80%, even more preferably at least about 85% or 90%homologous to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions tothe sequence of SEQ ID NO:1, 3, 10, or 11 corresponds to anaturally-occurring nucleic acid molecule.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein). In addition to the B7-4 orPD-1 nucleotide sequences shown in SEQ ID NO:1, 3, 10, and 11, it shouldbe appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to minor changes in the nucleotide or amino acidsequences of a B7-4 or PD-1 may exist within a population. Such geneticpolymorphism in a B7-4 or PD-1 gene may exist among individuals within apopulation due to natural allelic variation. Such natural allelicvariations can typically result in 1-2% variance in the nucleotidesequence of the gene. Such nucleotide variations and resulting aminoacid polymorphisms in a B7-4 or PD-1 that are the result of naturalallelic variation and that do not alter the functional activity of aB7-4 or PD-1 polypeptide are within the scope of the invention.

In addition to naturally-occurring allelic variants of B7-4 or PD-1sequences that may exist in the population, the skilled artisan willfurther appreciate that minor changes may be introduced by mutation intonucleotide sequences, e.g., of SEQ ID NO:1, 3, 10, or 11, therebyleading to changes in the amino acid sequence of the encoded protein,without altering the functional activity of a B7-4 or PD-1 protein. Forexample, nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues may be made in the sequence of SEQID NO:1, 3, 10, or 11. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence of a B7-4 nucleic acidmolecule (e.g., the sequence of SEQ ID NO:1, 3, 10, or 11) withoutaltering the functional activity of a B7-4 or PD-1 molecule. Preferably,residues in the extracellular domain of B7-4 or PD-1 which are found tobe required for binding of B7-4 to a receptor or PD-1 to a naturalligand (e.g., identified using an alanine scanning mutagenesis screen orother art recognized screening assay) are not altered. For B7-4molecules, exemplary residues which are non-essential and, therefore,amenable to substitution, can be identified by one of ordinary skill inthe art by performing an amino acid alignment of B7 family members (orof B7-4 family members) and determining residues that are not conserved.Such residues, because they have not been conserved, are more likelyamenable to substitution.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding B7-4 or PD-1 proteins that contain changes in aminoacid residues that are not essential for a B7-4 or PD-1 activity. SuchB7-4 or PD-1 proteins differ in amino acid sequence from SEQ ID NO:2, 4,or 12 yet retain an inherent B7-4 activity or, in the case of PD-1,retain the ability to bind to B7-4. An isolated nucleic acid moleculeencoding a non-natural variant of a B7-4 or PD-1 protein can be createdby introducing one or more nucleotide substitutions, additions ordeletions into the nucleotide sequence of SEQ ID NO:1, 3, 10, or 11 suchthat one or more amino acid substitutions, additions or deletions areintroduced into the encoded protein. Mutations can be introduced intoSEQ ID NO:1, 3, 10, or 11 by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. Preferably, conservative aminoacid substitutions are made at one or more non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art, including basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in a B7-4 or PD-1 is preferably replaced with another amino acidresidue from the same side chain family.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a B7-4 or PD-1 coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened fortheir ability to bind to DNA and/or activate transcription, to identifymutants that retain functional activity. Following mutagenesis, theencoded B7-4 or PD-1 mutant protein can be expressed recombinantly in ahost cell and the functional activity of the mutant protein can bedetermined using assays available in the art for assessing a B7-4 orPD-1 activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding B7-4 or PD-1 proteins that contain changes in aminoacid residues that are not essential for activity.

Yet another aspect of the invention pertains to isolated nucleic acidmolecules encoding a B7-4 or PD-1 fusion proteins. Such nucleic acidmolecules, comprising at least a first nucleotide sequence encoding aB7-4 or PD-1 protein, polypeptide or peptide operatively linked to asecond nucleotide sequence encoding a non- a B7-4 or PD-1 protein,polypeptide or peptide, can be prepared by standard recombinant DNAtechniques.

In a preferred embodiment, a mutant B7-4 protein can be assayed for theability to: 1) costimulate (or inhibit the costimulation of, e.g., insoluble form) the proliferation and/or effector function of activatedimmune cells; 2) bind to an anti-B7 family- or anti B7-4-antibody;and/or 3) bind to a natural receptor(s) of B7-4 (e.g., PD-1).

In a preferred embodiment, a mutant PD-1 protein can be assayed for theability to: 1) inhibit the costimulation of (e.g., in soluble form) theproliferation and/or effector function of activated immune cells; 2)bind to an anti-PD-1 antibody; and/or 3) bind to a natural ligand(s) ofPD-1 (e.g., B7-4).

In addition to the nucleic acid molecules encoding B7-4 or PD-1 proteinsdescribed above, isolated nucleic acid molecules which are antisensethereto can be used as modulating agents. An “antisense” nucleic acidcomprises a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. Accordingly, an antisense nucleic acid can hydrogen bond to asense nucleic acid. The antisense nucleic acid can be complementary toan entire B7-4 or PD-1 coding strand, or only to a portion thereof. Inone embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence encodingB7-4 or PD-1. The term “coding region” refers to the region of thenucleotide sequence comprising codons which are translated into aminoacid residues. In another embodiment, the antisense nucleic acidmolecule is antisense to a “noncoding region” of the coding strand of anucleotide sequence encoding B7-4 or PD-1. The term “noncoding region”refers to 5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

Given the coding strand sequences encoding B7-4 or PD-1 disclosedherein, antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof B7-4 or PD-1 mRNA, but more preferably is an oligonucleotide which isantisense to only a portion of the coding or noncoding region of B7-4 orPD-1 mRNA. For example, the antisense oligonucleotide can becomplementary to the region surrounding the translation start site ofB7-4 or PD-1 mRNA. An antisense oligonucleotide can be, for example,about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Anantisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid molecule (e.g.,an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid is of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a B7-4 or PD-1protein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention include direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid molecule of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandednucleic acid molecule, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haseloff and Gerlach (1988) Nature 334:585-591)) can beused to catalytically cleave B7-4 or PD-1 mRNA transcripts to therebyinhibit translation of B7-4 or PD-1 mRNA. A ribozyme having specificityfor a B7-4 or PD-1-encoding nucleic acid can be designed based upon thenucleotide sequence of a B7-4 or PD-1 cDNA disclosed herein (i.e., SEQID NO:1, 3, 10, or 11). For example, a derivative of a Tetrahymena L-19IVS RNA can be constructed in which the nucleotide sequence of theactive site is complementary to the nucleotide sequence to be cleaved ina B7-4 or PD-1-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No.4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, B7-4or PD-1 mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Bartel,D. and Szostak, J. W. (1993) Science 261:1411-1418.

Alternatively, B7-4 or PD-1 gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofthe B7-4 or PD-1 (e.g., the B7-4 or PD-1 promoter and/or enhancers) toform triple helical structures that prevent transcription of the B7-4 orPD-1 gene in target cells. See generally, Helene, C. (1991) AnticancerDrug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci.660:27-36; and Maher, L. J. (1992) Bioessays 14(12):807-15.

In yet another embodiment, the B7-4 or PD-1 nucleic acid molecules ofthe present invention can be modified at the base moiety, sugar moiety,or phosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acid molecules can be modified to generatepeptide nucleic acids (see Hyrup, B. and Nielsen, P. E. (1996) Bioorg.Med Chem. 4(1):5-23). As used herein, the terms “peptide nucleic acids”or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs has been shown to allow for specific hybridization to DNA andRNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup and Nielsen (1996) supra andPerry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs of B7-4 or PD-1 nucleic acid molecules can be used in therapeuticand diagnostic applications. For example, PNAs can be used as antisenseor antigene agents for sequence-specific modulation of gene expressionby, for example, inducing transcription or translation arrest orinhibiting replication. PNAs of B7-4 or PD-1 nucleic acid molecules canalso be used in the analysis of single base pair mutations in a gene,(e.g., by PNA-directed PCR clamping); as ‘artificial restrictionenzymes’ when used in combination with other enzymes, (e.g., S1nucleases (Hyrup and Nielsen (1996) supra)); or as probes or primers forDNA sequencing or hybridization (Hyrup B. and Nielsen (1996) supra;Perry-O'Keefe et al. (1996) supra).

In another embodiment, PNAs of B7-4 or PD-1 can be modified, (e.g., toenhance their stability or cellular uptake), by attaching lipophilic orother helper groups to PNA, by the formation of PNA-DNA chimeras, or bythe use of liposomes or other techniques of drug delivery known in theart. For example, PNA-DNA chimeras of B7-4 or PD-1 nucleic acidmolecules can be generated which may combine the advantageous propertiesof PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g.,RNAse H and DNA polymerases), to interact with the DNA portion while thePNA portion would provide high binding affinity and specificity. PNA-DNAchimeras can be linked using linkers of appropriate lengths selected interms of base stacking, number of bonds between the nucleobases, andorientation (Hyrup B. and Nielsen (1996) supra). The synthesis ofPNA-DNA chimeras can be performed as described in Hyrup B. and Nielsen(1996) supra and Finn P. J. et al. (1996) Nucleic Acids Res.24(17):3357-63. For example, a DNA chain can be synthesized on a solidsupport using standard phosphoramidite coupling chemistry. Modifiednucleoside analogs, (e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite), can be used as a linker between the PNA and the 5′ endof DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17:5973-88). PNAmonomers are then coupled in a stepwise manner to produce a chimericmolecule with a 5′ PNA segment and a 3′ DNA segment (Finn P. J. et al.(1996) supra). Alternatively, chimeric molecules can be synthesized witha 5′ DNA segment and a 3′ PNA segment (Peterser, K. H. et al. (1975)Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al. (1988) Biotechniques 6:958-976) or intercalating agents. (See,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide can be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

III. Isolated B7-4 or PD-1 Proteins and Anti-B7-4 or PD-1 Antibodies

In addition, isolated B7-4 or PD-1 proteins, and biologically activeportions thereof, as well as anti-B7-4 or PD-1 antibodies can be used asmodulating agents. In one embodiment, native B7-4 or PD-1 proteins canbe isolated from cells or tissue sources by an appropriate purificationscheme using standard protein purification techniques. In anotherembodiment, B7-4 or PD-1 proteins are produced by recombinant DNAtechniques. Alternative to recombinant expression, a B7-4 or PD-1protein or polypeptide can be synthesized chemically using standardpeptide synthesis techniques.

Another aspect of the invention pertains to isolated B7-4 or PD-1proteins. Preferably, the B7-4 or PD-1 proteins comprise the amino acidsequence encoded by SEQ ID NO:1, 3, 10 or 11. In another preferredembodiment, the protein comprises the amino acid sequence of SEQ IDNO:2, 4, or 12. In other embodiments, the protein has at least 50%, atleast 60% amino acid identity, more preferably 70% amino acid identity,more preferably 80%, and even more preferably, 90% or 95% amino acididentity with the amino acid sequence shown in SEQ ID NO:2, 4, or 12.

In other embodiments, the invention provides isolated portions of a B7-4or PD-1 protein. For example, B7-4 proteins comprise a signal sequence,and an IgV domain and an IgC domain. The signal sequence of SEQ ID NO:2is shown from about amino acid 1 to about amino acid 18. The signalsequence of SEQ ID NO:4 is shown from about amino acid 1 to about aminoacid 18. The IgV domain of SEQ ID NO:2 is shown from about amino acid 19to about amino acid 134 and the IgV domain of SEQ ID NO:4 is shown fromabout amino acid 19 to about amino acid 134. The IgC domain of SEQ IDNO:2 is shown from about amino acid 135 to about amino acid 227 and theIgC domain of SEQ ID NO:4 is shown from about amino acid 135 to aboutamino acid 227. The hydrophilic tail of the B7-4 molecule exemplified inSEQ ID NO:2 comprises a hydrophilic tail shown from about amino acid 228to about amino acid 245. The B7-4 polypeptide exemplified in SEQ ID NO:4comprises a transmembrane domain shown from about amino acid 239 toabout amino acid 259 of SEQ ID NO:4 and a cytoplasmic domain shown fromabout amino acid 260 to about amino acid 290 of SEQ ID NO:4. The PD-1polypeptide is 288 amino acids in length and its domain structure isknown in the art (Shinohara et al. (1994) Genomics 23:704). Thepredicted mature form of the protein contains about 268 amino acids andcomprises an extracellular domain (147 amino acids), a transmembranedomain (27 amino acids), a transmembrane region (27 amino acids) and acytoplasmic domain (94 amino acids) Four potential N-glycosylation sitesare found in the extracellular domain (U.S. Pat. No. 5,698,520). The 68amino acid residues between two cysteine residues (cys 54 and cys 123)bear resemblance to a disulfide-linked immunoglobulin domain of theV-set sequences (U.S. Pat. No. 5,698,520).

The invention further pertains to soluble forms of B7-4 or PD-1proteins. Such forms can be naturally occurring, e.g., as shown in SEQID NO:2 or can be engineered and can comprise, e.g., an extracellulardomain of a B7-4 or PD-1 protein. Exemplary B7-4 extracellular domainscomprise from about amino acids 19-238 of SEQ ID NO:4. Exemplary PD-1extracellular domains comprise from about amino acids 21-288 of SEQ IDNO:12.

In one embodiment, the extracellular domain of a B7-4 polypeptidecomprises the mature form of a B7-4 polypeptide, e.g., the IgV and IgCdomains, but not the transmembrane and cytoplasmic domains of a B7-4polypeptide (e.g., from about amino acid 19 to amino acid 238 of SEQ IDNO:4) or from about amino acid 19 to amino acid 245 of SEQ. ID. NO:2.

In one embodiment, the extracellular domain of a PD-1 polypeptidecomprises the mature form of a PD-1 polypeptide, e.g., immunoglobulinsuperfamily domains (e.g., V-set sequences), but not the transmembraneand cytoplasmic domains of a PD-1 polypeptide (e.g., from about aminoacid 21-288 of SEQ ID NO:12).

Biologically active portions of a B7-4 or PD-1 protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the B7-4 or PD-1 protein, which includeless amino acids than the full length B7-4 or PD-1 proteins, and exhibitat least one activity of a B7-4 or PD-1 protein, preferably the abilityto bind to a natural binding partner. Typically, biologically activeportions comprise a domain or motif with at least one activity of theB7-4 or PD-1 protein. A biologically active portion of a B7-4 or PD-1protein can be a polypeptide which is, for example, at least 10, 25, 50,100, 150, 200 or more amino acids in length.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence. The residues at corresponding positions are then compared andwhen a position in one sequence is occupied by the same residue as thecorresponding position in the other sequence, then the molecules areidentical at that position. The percent identity between two sequences,therefore, is a function of the number of identical positions shared bytwo sequences (i.e., % identity=# of identical positions/total # ofpositions×100). The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.As used herein amino acid or nucleic acid “identity” is equivalent toamino acid or nucleic acid “homology”.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the GAP program in the GCGsoftware package (available at the Genetics Computer Group website),using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.In yet another preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (available at the Genetics Computer Group website),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to B7-4 or PD-1 nucleic acid molecules of the invention.BLAST protein searches can be performed with the XBLAST program,score=50, wordlength=3 to obtain amino acid sequences homologous to B7-4or PD-1 protein molecules of the invention. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. For example,the nucleotide sequences of the invention were analyzed using thedefault Blastn matrix 1-3 with gap penalties set at: existence 11 andextension 1. The amino acid sequences of the invention were analyzedusing the default settings: the Blosum 62 matrix with gap penalties setat existence 11 and extension 1. See the NCBI website.

The invention also provides B7-4 or PD-1 chimeric or fusion proteins. Asused herein, a B7-4 or PD-1 “chimeric protein” or “fusion protein”comprises a B7-4 or PD-1 polypeptide operatively linked to a non-B7-4 orPD-1 polypeptide. A “B7-4 or PD-1 polypeptide” refers to a polypeptidehaving an amino acid sequence corresponding to B7-4 or PD-1 polypeptide,whereas a “non-B7-4 or PD-1 polypeptide” refers to a polypeptide havingan amino acid sequence corresponding to a protein which is notsubstantially homologous to the B7-4 or PD-1 protein, e.g., a proteinwhich is different from the B7-4 or PD-1 protein and which is derivedfrom the same or a different organism. Within a B7-4 or PD-1 fusionprotein the B7-4 or PD-1 polypeptide can correspond to all or a portionof a B7-4 or PD-1 protein. In a preferred embodiment, a B7-4 or PD-1fusion protein comprises at least one biologically active portion of aB7-4 or PD-1 protein, e.g., an extracellular domain of a B7-4 or PD-1protein. Within the fusion protein, the term “operatively linked” isintended to indicate that the B7-4 or PD-1 polypeptide and the non-B7-4or PD-1 polypeptide are fused in-frame to each other. The non-B7-4 orPD-1 polypeptide can be fused to the N-terminus or C-terminus of theB7-4 or PD-1 polypeptide.

For example, in one embodiment, the fusion protein is a GST-B7-4 orGST-PD-1 fusion protein in which the B7-4 or PD-1 sequences are fused tothe C-terminus of the GST sequences. In another embodiment, the fusionprotein is a B7-4 or PD-1-HA fusion protein in which the B7-4 or PD-1nucleotide sequence is inserted in a vector such as pCEP4-HA vector(Herrscher, R. F. et al. (1995) Genes Dev. 9:3067-3082) such that theB7-4 or PD-1 sequences are fused in frame to an influenza hemagglutininepitope tag. Such fusion proteins can facilitate the purification of arecombinant B7-4 or PD-1 protein.

A B7-4 or PD-1 fusion protein can be produced by recombinant expressionof a nucleotide sequence encoding a first peptide having B74 activityand a nucleotide sequence encoding second peptide corresponding to amoiety that alters the solubility, affinity, stability or valency of thefirst peptide, for example, an immunoglobulin constant region.Preferably, the first peptide consists of a portion of the B7-4polypeptide (e.g., a portion of amino acid residues 1-238 or 19-238(after cleavage of the signal sequence) of the sequence shown in SEQ IDNO:4 that is sufficient to modulate costimulation or inhibition ofactivated immune cells). In another preferred embodiment, the firstpeptide consists of a portion of a PD-1 polypeptide (e.g., a portion ofamino acid residues 1-288 (or 21-288 after cleavage of the signalpeptide) of the sequence shown in SEQ ID NO:12 that is sufficient tomodulate costimulation or inhibition of activated immune cells) Thesecond peptide can include an immunoglobulin constant region, forexample, a human Cγ1 domain or Cγ4 domain (e.g., the hinge, CH2 and CH3regions of human IgCγ1, or human IgCγ4, see e.g., Capon et al. U.S. Pat.Nos. 5,116,964; 5,580,756; 5,844,095 and the like, incorporated hereinby reference). A resulting fusion protein may have altered B7-4 or PD-1solubility, binding affinity, stability and/or valency (i.e., the numberof binding sites available per molecule) and may increase the efficiencyof protein purification. Fusion proteins and peptides produced byrecombinant techniques can be secreted and isolated from a mixture ofcells and medium containing the protein or peptide. Alternatively, theprotein or peptide can be retained cytoplasmically and the cellsharvested, lysed and the protein isolated. A cell culture typicallyincludes host cells, media and other byproducts. Suitable media for cellculture are well known in the art. Protein and peptides can be isolatedfrom cell culture media, host cells, or both using techniques known inthe art for purifying proteins and peptides. Techniques for transfectinghost cells and purifying proteins and peptides are known in the art.

Particularly preferred B7-4 or PD-1 Ig fusion proteins include theextracellular domain portion or variable region-like domain of a humanB7-4 or PD-1 coupled to an immunoglobulin constant region (e.g., the Fcregion). The immunoglobulin constant region may contain geneticmodifications which reduce or eliminate effector activity inherent inthe immunoglobulin structure. For example, DNA encoding theextracellular portion of a B7-4 or PD-1 polypeptide can be joined to DNAencoding the hinge, CH2 and CH3 regions of human IgGγ1 and/or IgGγ4modified by site directed mutagenesis, e.g., as taught in WO 97/28267.

Preferably, a B7-4 or PD-1 fusion protein of the invention is producedby standard recombinant DNA techniques. For example, DNA fragmentscoding for the different polypeptide sequences are ligated togetherin-frame in accordance with conventional techniques, for exampleemploying blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and reamplified to generate a chimeric genesequence (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. John Wiley & Sons: 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST polypeptide or an HA epitope tag). A B7-4 or PD-1 encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the B7-4 or PD-1 protein.

In another embodiment, the fusion protein is a B7-4 or PD-1 proteincontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofB7-4 or PD-1 can be increased through use of a heterologous signalsequence.

The B7-4 or PD-1 fusion proteins of the invention can be incorporatedinto pharmaceutical compositions and administered to a subject in vivo.Use of B7-4 or PD-1 fusion proteins is useful therapeutically for thetreatment of immunological disorders, e.g., autoimmune diseases, or inthe case of inhibiting rejection of transplants. Moreover, the B7-4 orPD-1-fusion proteins of the invention can be used as immunogens toproduce anti-B7-4 or PD-1 antibodies in a subject, to purify B7-4 orPD-1 and in screening assays to identify molecules which inhibit theinteraction of B7-4 with a B7-4 receptor, e.g., PD-1.

Preferably, a B7-4 or PD-1 chimeric or fusion protein of the inventionis produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A B7-4 orPD-1-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the B7-4 or PD-1protein.

The present invention also pertains to variants of the B7-4 or PD-1proteins which function as either B7-4 or PD-1 agonists (mimetics) or asB7-4 or PD-1 antagonists. Variants of the B7-4 or PD-1 proteins can begenerated by mutagenesis, e.g., discrete point mutation or truncation ofa B7-4 or PD-1 protein. An agonist of the B7-4 or PD-1 proteins canretain substantially the same, or a subset, of the biological activitiesof the naturally occurring form of a B7-4 or PD-1 protein. An antagonistof a B7-4 or PD-1 protein can inhibit one or more of the activities ofthe naturally occurring form of the B7-4 or PD-1 protein by, forexample, competitively modulating a cellular activity of a B7-4 or PD-1protein. Thus, specific biological effects can be elicited by treatmentwith a variant of limited function. In one embodiment, treatment of asubject with a variant having a subset of the biological activities ofthe naturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of theB7-4 or PD-1 protein.

In one embodiment, variants of a B7-4 or PD-1 protein which function aseither B7-4 or PD-1 agonists (mimetics) or as B7-4 or PD-1 antagonistscan be identified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of a B7-4 or PD-1 protein for B7-4 or PD-1 proteinagonist or antagonist activity. In one embodiment, a variegated libraryof B7-4 or PD-1 variants is generated by combinatorial mutagenesis atthe nucleic acid level and is encoded by a variegated gene library. Avariegated library of B7-4 or PD-1 variants can be produced by, forexample, enzymatically ligating a mixture of synthetic oligonucleotidesinto gene sequences such that a degenerate set of potential B7-4 or PD-1sequences is expressible as individual polypeptides, or alternatively,as a set of larger fusion proteins (e.g., for phage display) containingthe set of B7-4 or PD-1 sequences therein. There are a variety ofmethods which can be used to produce libraries of potential B7-4 or PD-1variants from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be performed in an automatic DNAsynthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential B7-4 or PD-1 sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (I984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477.

In addition, libraries of fragments of a B7-4 or PD-1 protein codingsequence can be used to generate a variegated population of B7-4 or PD-1fragments for screening and subsequent selection of variants of a B7-4or PD-1 protein. In one embodiment, a library of coding sequencefragments can be generated by treating a double stranded PCR fragment ofa B7-4 or PD-1 coding sequence with a nuclease under conditions whereinnicking occurs only about once per molecule, denaturing the doublestranded DNA, renaturing the DNA to form double stranded DNA which caninclude sense/antisense pairs from different nicked products, removingsingle stranded portions from reformed duplexes by treatment with S1nuclease, and ligating the resulting fragment library into an expressionvector. By this method, an expression library can be derived whichencodes N-terminal, C-terminal and internal fragments of various sizesof the B7-4 or PD-1 protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of B7-4 or PD-1 proteins. Themost widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify B7-4 or PD-1 variants (Arkin and Youvan (1992) Proc. Natl.Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993) Protein Eng.6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated B7-4 or PD-1 library. For example, a library of expressionvectors can be transfected into a cell line which ordinarily synthesizesand secretes B7-4 or PD-1. The transfected cells are then cultured suchthat B7-4 or PD-1 and a particular mutant B7-4 or PD-1 are secreted andthe effect of expression of the mutant on B7-4 or PD-1 activity in cellsupernatants can be detected, e.g., by any of a number of functionalassays. Plasmid DNA can then be recovered from the cells which score forinhibition, or alternatively, potentiation of B7-4 or PD-1 activity, andthe individual clones further characterized.

In addition to B7-4 or PD-1 polypeptides consisting only ofnaturally-occurring amino acids, B7-4 or PD-1 peptidomimetics are alsoprovided. Peptide analogs are commonly used in the pharmaceuticalindustry as non-peptide drugs with properties analogous to those of thetemplate peptide. These types of non-peptide compound are termed“peptide mimetics” or “peptidomimetics” (Fauchere, J. (I986) Adv. DrugRes. 15:29; Veber and Freidinger (1985) TINS p.392; and Evans et al.(1987) J. Med. Chem. 30:1229, which are incorporated herein byreference) and are usually developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides can be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biological or pharmacological activity), such as human B7-4 orPD-1, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH2NH—, —CH2S—,—CH2—CH2—, —CH═CH— (cis and trans), —COCH2—, —CH(OH)CH2—, and —CH2SO—,by methods known in the art and further described in the followingreferences: Spatola, A. F. in “Chemistry and Biochemistry of AminoAcids, Peptides, and Proteins” Weinstein, B., ed., Marcel Dekker, NewYork, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1,Issue 3, “Peptide Backbone Modifications” (general review); Morley, J.S. (1980) Trends Pharm. Sci. pp. 463-468 (general review); Hudson, D. etal. (1979) Int. J. Pept. Prot. Res. 14:177-185 (—CH2NH—, CH2CH2—);Spatola, A. F. et al. (1986) Life Sci. 38:1243-1249 (—CH2—S); Hann, M.M. (1982) J. Chem. Soc. Perkin Trans. I. 307-314 (—CH—CH—, cis andtrans); Almquist, R. G. et al. (190) J. Med. Chem. 23:1392-1398(—COCH2—); Jennings-White, C. et al. (1982) Tetrahedron Lett. 23:2533(—COCH2—); Szelke, M. et al. European Appln. EP 45665 (1982) CA:97:39405 (1982)(—CH(OH)CH2—); Holladay, M. W. et al. (1983) TetrahedronLett. (1983) 24:4401-4404 (—C(OH)CH2—); and Hruby, V. J. (1982) LifeSci. (1982) 31:189-199 (—CH2—S—); each of which is incorporated hereinby reference. A particularly preferred non-peptide linkage is —CH2NH—.Such peptide mimetics may have significant advantages over polypeptideembodiments, including, for example: more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers. Labeling of peptidomimetics usually involves covalent attachmentof one or more labels, directly or through a spacer (e.g., an amidegroup), to non-interfering position(s) on the peptidomimetic that arepredicted by quantitative structure-activity data and/or molecularmodeling. Such non-interfering positions generally are positions that donot form direct contacts with the macromolecules(s) to which thepeptidomimetic binds to produce the therapeutic effect. Derivitization(e.g., labeling) of peptidomimetics should not substantially interferewith the desired biological or pharmacological activity of thepeptidomimetic.

Systematic substitution of one or more amino acids of a B7-4 or PD-1amino acid sequence with a D-amino acid of the same type (e.g., D-lysinein place of L-lysine) can be used to generate more stable peptides. Inaddition, constrained peptides comprising a B7-4 or PD-1 amino acidsequence or a substantially identical sequence variation can begenerated by methods known in the art (Rizo and Gierasch (1992) Annu.Rev. Biochem. 61:387, incorporated herein by reference); for example, byadding internal cysteine residues capable of forming intramoleculardisulfide bridges which cyclize the peptide.

The amino acid sequences of B7-4 or PD-1 polypeptides identified hereinwill enable those of skill in the art to produce polypeptidescorresponding to B7-4 or PD-1 peptide sequences and sequence variantsthereof. Such polypeptides can be produced in prokaryotic or eukaryotichost cells by expression of polynucleotides encoding a B7-4 or PD-1peptide sequence, frequently as part of a larger polypeptide.Alternatively, such peptides can be synthesized by chemical methods.Methods for expression of heterologous proteins in recombinant hosts,chemical synthesis of polypeptides, and in vitro translation are wellknown in the art and are described further in Maniatis et al. MolecularCloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.;Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to MolecularCloning Techniques (1987), Academic Press, Inc., San Diego, Calif.;Merrifield, J. (1969) J. Am. Chem. Soc. 91:501; Chaiken I. M. (1981) CRCCrit. Rev. Biochem. 11: 255; Kaiser et al. (1989) Science 243:187;Merrifield, B. (1986) Science 232:342; Kent, S. B. H. (1988) Annu. Rev.Biochem. 57:957; and Offord, R. E. (1980) Semisynthetic Proteins, WileyPublishing, which are incorporated herein by reference).

Peptides can be produced, typically by direct chemical synthesis, andused e.g., as agonists or antagonists of a B7-4/PD-1 interaction.Peptides can be produced as modified peptides, with nonpeptide moietiesattached by covalent linkage to the N-terminus and/or C-terminus. Incertain preferred embodiments, either the carboxy-terminus or theamino-terminus, or both, are chemically modified. The most commonmodifications of the terminal amino and carboxyl groups are acetylationand amidation, respectively. Amino-terminal modifications such asacylation (e.g., acetylation) or alkylation (e.g., methylation) andcarboxy-terminal-modifications such as amidation, as well as otherterminal modifications, including cyclization, can be incorporated intovarious embodiments of the invention. Certain amino-terminal and/orcarboxy-terminal modifications and/or peptide extensions to the coresequence can provide advantageous physical, chemical, biochemical, andpharmacological properties, such as: enhanced stability, increasedpotency and/or efficacy, resistance to serum proteases, desirablepharmacokinetic properties, and others. Peptides can be usedtherapeutically to treat disease, e.g., by altering costimulation in apatient.

An isolated B7-4 or PD-1 protein, or a portion or fragment thereof (or anucleic acid molecule encoding such a polypeptide), can be used as animmunogen to generate antibodies that bind B7-4 or PD-1 using standardtechniques for polyclonal and monoclonal antibody preparation. Afull-length B7-4 or PD-1 protein can be used, or alternatively, theinvention provides antigenic peptide fragments of B7-4 or PD-1 for useas immunogens. The antigenic peptide of B7-4 or PD-1 comprises at least8 amino acid residues and encompasses an epitope of B7-4 or PD-1 suchthat an antibody raised against the peptide forms a specific immunecomplex with B7-4 or PD-1. Preferably, the antigenic peptide comprisesat least 10 amino acid residues, more preferably at least 15 amino acidresidues, even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues.

Alternatively, an antigenic peptide fragment of a B7-4 or PD-1polypeptide can be used as the immunogen. An antigenic peptide fragmentof a B7-4 or PD-1 polypeptide typically comprises at least 8 amino acidresidues of the amino acid sequence shown in SEQ ID NO:2, 4, or 12 andencompasses an epitope of a B7-4 or PD-1 polypeptide such that anantibody raised against the peptide forms an immune complex with a B7-4or PD-1 molecule. Preferred epitopes encompassed by the antigenicpeptide are regions of B7-4 or PD-1 that are located on the surface ofthe protein, e.g., hydrophilic regions. In one embodiment, an antibodybinds substantially specifically to a B7-4 or PD-1 molecule. In anotherembodiment, an antibody binds specifically to a B7-4 or PD-1polypeptide.

Preferably, the antigenic peptide comprises at least about 10 amino acidresidues, more preferably at least about 15 amino acid residues, evenmore preferably at least 20 about amino acid residues, and mostpreferably at least about 30 amino acid residues. Preferred epitopesencompassed by the antigenic peptide are regions of a B7-4 or PD-1polypeptide that are located on the surface of the protein, e.g.,hydrophilic regions, and that are unique to a B7-4 or PD-1 polypeptide.In one embodiment such epitopes can be specific for a B7-4 or PD-1proteins from one species, such as mouse or human (i.e., an antigenicpeptide that spans a region of a B7-4 or PD-1 polypeptide that is notconserved across species is used as immunogen; such non conservedresidues can be determined using an alignment such as that providedherein). A standard hydrophobicity analysis of the B7-4 or PD-1 proteincan be performed to identify hydrophilic regions.

A B7-4 or PD-1 immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, a recombinantly expressed B7-4 or PD-1 protein ora chemically synthesized B7-4 or PD-1 peptide. The preparation canfurther include an adjuvant, such as Freund's complete or incompleteadjuvant, or similar immunostimulatory agent. Immunization of a suitablesubject with an immunogenic B7-4 or PD-1 preparation induces apolyclonal anti- B7-4 or PD-1 antibody response.

In another embodiment, nucleic acid vaccines can be administered by avariety of means, for example, by injection (e.g., intramuscular,intradermal, or the biolistic injection of DNA-coated gold particlesinto the epidermis with a gene gun that uses a particle accelerator or acompressed gas to inject the particles into the skin (Haynes et al.1996. J. Biotechnol. 44:37)). Alternatively, nucleic acid vaccines canbe administered by non-invasive means. For example, pure orlipid-formulated DNA can be delivered to the respiratory system ortargeted elsewhere, e.g., Peyers patches by oral delivery of DNA(Schubbert. 1997. Proc. Natl. Acad. Sci. USA 94:961). Attenuatedmicroorganisms can be used for delivery to mucosal surfaces. (Sizemoreet al. 1995. Science. 270:29)

Polyclonal anti-B7-4 or PD-1 antibodies can be prepared as describedabove by immunizing a suitable subject with a B7-4 or PD-1 immunogen.The anti-B7-4 or PD-1 antibody titer in the immunized subject can bemonitored over time by standard techniques, such as with an enzymelinked immunosorbent assay (ELISA) using immobilized a B7-4 or PD-1polypeptide. If desired, the antibody molecules directed against a B7-4or PD-1 polypeptide can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti- B7-4 or PD-1 antibody titers arehighest, antibody-producing cells can be obtained from the subject andused to prepare monoclonal antibodies by standard techniques, such asthe hybridoma technique originally described by Kohler and Milstein(1975) Nature 256:495-497) (see also Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. 76:2927-31; and Yeh et al. (1982) Int. J.Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique(Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generallyKenneth, R. H. in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); Lerner, E. A.(1981) Yale J. Biol. Med. 54:387-402; Gefter, M. L. et al. (1977)Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a B7-4 or PD-1 immunogen as described above, and theculture supernatants of the resulting hybridoma cells are screened toidentify a hybridoma producing a monoclonal antibody that binds to aB7-4 or PD-1 polypeptide, preferably specifically.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-B7-4 or PD-1 monoclonal antibody (see, e.g., Galfre, G. et al.(1977) Nature 266:55052; Gefter et al. (1977) supra; Lerner (1981)supra; Kenneth (1980) supra). Moreover, the ordinary skilled worker willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line. Preferred immortal cell lines aremouse myeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from theAmerican Type Culture Collection (ATCC), Rockville, Md. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bind aB7-4 or PD-1 molecule, e.g., using a standard ELISA assay.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal anti-B7-4 or PD-1 antibody can be identified and isolatedby screening a recombinant combinatorial immunoglobulin library (e.g.,an antibody phage display library) with a B7-4 or PD-1 to therebyisolate immunoglobulin library members that bind a B7-4 or PD-1polypeptide. Kits for generating and screening phage display librariesare commercially available (e.g., the Pharmacia Recombinant PhageAntibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™Phage Display Kit, Catalog No. 240612). Additionally, examples ofmethods and reagents particularly amenable for use in generating andscreening antibody display library can be found in, for example, Ladneret al. U.S. Pat. No. 5,223,409; Kang et al. International PublicationNo. WO 92/18619; Dower et al. International Publication No. WO 91/17271;Winter et al. International Publication WO 92/20791; Markland et al.International Publication No. WO 92/15679; Breitling et al.International Publication WO 93/01288; McCafferty et al. InternationalPublication No. WO 92/01047; Garrard et al. International PublicationNo. WO 92/09690; Ladner et al. International Publication No. WO90/02809; Fuchs et al. (1991) Biotechnology (NY) 9:1369-1372; Hay et al.(1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins etal. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580;Garrard et al. (1991) Biotechnology (NY) 9:1373-1377; Hoogenboom et al.(1991) Nucleic Acids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl.Acad. Sci. USA 88:7978-7982; and McCafferty et al. (1990) Nature348:552-554.

Additionally, recombinant anti-B7-4 or PD-1 antibodies, such as chimericand humanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Patent Publication PCT/US86/02269; Akira et al. EuropeanPatent Application 184,187; Taniguchi, M. European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT Application WO 86/01533; Cabilly et al. U.S. Pat. No.4,816,567; Cabilly et al. European Patent Application 125,023; Better etal. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad.Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sunet al. (1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987)Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shawet al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L.(1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214;Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525;Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J.Immunol. 141:4053-4060.

In addition, humanized antibodies can be made according to standardprotocols such as those disclosed in U.S. Pat. No. 5,565,332. In anotherembodiment, antibody chains or specific binding pair members can beproduced by recombination between vectors comprising nucleic acidmolecules encoding a fusion of a polypeptide chain of a specific bindingpair member and a component of a replicable geneic display package andvectors containing nucleic acid molecules encoding a second polypeptidechain of a single binding pair member using techniques known in the art,e.g., as described in U.S. Pat. Nos. 5,565,332, 5,871,907, or 5,733,743.The use of intracellular antibodies to inhibit protein function in acell is also known in the art (see e.g., Carlson, J. R. (1988) Mol.Cell. Biol. 8:2638-2646; Biocca, S. et al. (1990) EMBO J. 9:101-108;Werge, T. M. et al. (1990) FEBS Lett. 274:193-198; Carlson, J. R. (1993)Proc. Natl. Acad. Sci. USA 90:7427-7428; Marasco, W. A. et al. (1993)Proc. Natl. Acad. Sci. USA 90:7889-7893; Biocca, S. et al. (1994)Biotechnology (NY) 12:396-399; Chen, S-Y. et al. (1994) Hum. Gene Ther.5:595-601; Duan, L et al. (1994) Proc. Natl. Acad. Sci. USA91:5075-5079; Chen, S-Y. et al. (1994) Proc. Natl. Acad. Sci. USA91:5932-5936; Beerli, R. R. et al. (1994) J. Biol. Chem.269:23931-23936; Beerli, R. R. et al. (1994) Biochem. Biophys. Res.Commun. 204:666-672; Mhashilkar, A. M. et al. (1995) EMBO J.14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad. Sci. USA92:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al.).

In one embodiment, an antibody for use in the instant invention is abispecific antibody. A bispecific antibody has binding sites for twodifferent antigens within a single antibody molecule. Antigen bindingmay be simultaneous or sequential. Triomas and hybrid hybridomas are twoexamples of cell lines that can secrete bispecific antibodies. Examplesof bispecific antibodies produced by a hybrid hybridoma or a trioma aredisclosed in U.S. Pat. No. 4,474,893. Bispecific antibodies have beenconstructed by chemical means (Staerz et al. (1985) Nature 314:628, andPerez et al. (1985) Nature 316:354) and hybridoma technology (Staerz andBevan (1986) Proc. Natl. Acad. Sci. USA, 83:1453, and Staerz and Bevan(1986) Immunol. Today 7:241). Bispecific antibodies are also describedin U.S. Pat. No. 5,959,084. Fragments of bispecific antibodies aredescribed in U.S. Pat. No. 5,798,229.

Bispecific agents can also be generated by making heterohybridomas byfusing hybridomas or other cells making different antibodies, followedby identification of clones producing and co-assembling both antibodies.They can also be generated by chemical or genetic conjugation ofcomplete immunoglobulin chains or portions thereof such as Fab and Fvsequences. The antibody component can bind to PD-1 or B7-4.

An anti-B7-4 or PD-1 antibody (e.g., monoclonal antibody) can be used toisolate a B7-4 or PD-1 polypeptide by standard techniques, such asaffinity chromatography or immunoprecipitation. Anti-B7-4 or PD-1antibodies can facilitate the purification of natural B7-4 or PD-1polypeptides from cells and of recombinantly produced B7-4 or PD-1polypeptides expressed in host cells. Moreover, an anti-B7-4 or PD-1antibody can be used to detect a B7-4 or PD-1 protein (e.g., in acellular lysate or cell supernatant). Detection can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Accordingly, in one embodiment, an anti-B7-4 or PD-1 antibodyof the invention is labeled with a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidinibiotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S, and ³H.

Yet another aspect of the invention pertains to anti-B7-4 or PD-1antibodies that are obtainable by a process comprising:

(a) immunizing an animal with an immunogenic B7-4 or PD-1 protein, or animmunogenic portion thereof unique to a B7-4 or PD-1 polypeptide; and

(b) isolating from the animal antibodies that specifically bind to aB7-4 or PD-1 protein.

IV. Recombinant Expression Vectors and Host Cells

Nucleic acid molecules encoding a B7-4 or PD-1 family protein (or aportion thereof) can be contained in vectors, preferably expressionvectors. As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

Recombinant expression vectors can comprise a nucleic acid molecule ofthe invention in a form suitable for expression, e.g., constitutive orinducible expression, of a PD-1 or B7-4 molecule in the indicatorcell(s) of the nucleic acid in a host cell, which means that therecombinant expression vectors include one or more regulatory sequences,selected on the basis of the host cells to be used for expression, whichis operatively linked to the nucleic acid sequence to be expressed.Within a recombinant expression vector, “operably linked” is intended tomean that the nucleotide sequence of interest is linked to theregulatory sequence(s) in a manner which allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” includes promoters, enhancers andother expression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel (1990)Methods Enzymol. 185:3-7. Regulatory sequences include those whichdirect constitutive expression of a nucleotide sequence in many types ofhost cell and those which direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).It should be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,and the like. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein (e.g., B7-4 or PD-1 family proteins, mutant forms of B7-4 or PD-1proteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of B7-4 or PD-1 proteins in prokaryotic or eukaryotic cells.For example, B7-4 or PD-1 proteins can be expressed in bacterial cellssuch as E. coli, insect cells (using baculovirus expression vectors)yeast cells or mammalian cells. Suitable host cells are discussedfurther in Goeddel (1990) supra. Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Purified fusion proteins can be utilized in B7-4 or PD-1 activityassays, (e.g., direct assays or competitive assays described in detailbelow), or to generate antibodies specific for B7-4 or PD-1 proteins,for example.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al. (1990) Methods Enzymol. 185:60-89). Target gene expression fromthe pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S. (1990)Methods Enzymol. 185:119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the B7-4 or PD-1 expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerevisiae include pYepSec1 (Baldari, et al. (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz etal. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, a B7-4 or PD-1 polypeptide can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow, V. A. and Summers, M. D.(1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pMex-NeoI, pCDM8 (Seed, B. (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.et al. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Baneji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

Moreover, inducible regulatory systems for use in mammalian cells areknown in the art, for example systems in which gene expression isregulated by heavy metal ions (see e.g., Mayo et al. (1982) Cell29:99-108; Brinster et al. (1982) Nature 296:39-42; Searle et al. (1985)Mol. Cell. Biol. 5:1480-1489), heat shock (see e.g., Nouer et al. (1991)in Heat Shock Response, ed. Nouer, L., CRC, Boca Raton, Fla.,pp167-220), hormones (see e.g., Lee et al. (1981) Nature 294:228-232;Hynes et al. (1981) Proc. Natl. Acad. Sci. USA 78:2038-2042; Klock etal. (1987) Nature 329:734-736; Israel and Kaufman (1989) Nucl. AcidsRes. 17:2589-2604; and PCT Publication No. WO 93/23431), FK506-relatedmolecules (see e.g., PCT Publication No. WO 94/18317) or tetracyclines(Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; PCTPublication No. WO 94/29442; and PCT Publication No. WO 96/01313).Accordingly, in another embodiment, the invention provides a recombinantexpression vector in which a B7-4 or PD-1 DNA is operatively linked toan inducible eukaryotic promoter, thereby allowing for inducibleexpression of a B7-4 or PD-1 protein in eukaryotic cells.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to B7-4 or PD-1 mRNA. Regulatory sequencesoperatively linked to a nucleic acid cloned in the antisense orientationcan be chosen which direct the continuous expression of the antisenseRNA molecule in a variety of cell types, for instance viral promotersand/or enhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal. (1986) “Antisense RNA as a molecular tool for genetic analysis”Reviews—Trends in Genetics, Vol. 1(1).

The invention further pertains to host cells into which a recombinantexpression vector of the invention has been introduced. The terms “hostcell” and “recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aB7-4 or PD-1 protein can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells are known tothose skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a B7-4 or PD-1 protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a B7-4 or PD-1protein. Accordingly, the invention further provides methods forproducing a B7-4 or PD-1 protein using the host cells of the invention.In one embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding a B7-4 orPD-1 protein has been introduced) in a suitable medium such that a B7-4or PD-1 protein is produced. In another embodiment, the method furthercomprises isolating a B7-4 or PD-1 protein from the medium or the hostcell.

Certain host cells can also be used to produce non-human transgenicanimals. For example, in one embodiment, a host cell is a fertilizedoocyte or an embryonic stem cell into which B7-4 or PD-1-codingsequences have been introduced. Such host cells can then be used tocreate non-human transgenic animals in which exogenous B7-4 or PD-1sequences have been introduced into their genome or homologousrecombinant animals in which endogenous B7-4 or PD-1 sequences have beenaltered. Such animals are useful for studying the function and/oractivity of a B7-4 or PD-1 polypeptide and for identifying and/orevaluating modulators of B7-4 or PD-1 activity. As used herein, a“transgenic animal” is a non-human animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include non-human primates, sheep, dogs, cows, goats, chickens,amphibians, and the like. A transgene is exogenous DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous B7-4 or PD-1 gene has beenaltered by homologous recombination between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal.

A transgenic animal can be created by introducing a B7-4 orPD-1-encoding nucleic acid molecule into the male pronucleus of afertilized oocyte, e.g., by microinjection, retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The B7-4 or PD-1 cDNA sequence of SEQ ID NO:1, 3, 10, or 11 can beintroduced as a transgene into the genome of a non-human animal.Alternatively, a nonhuman homologue of a human B7-4 or PD-1 gene, suchas a mouse or rat B7-4 or PD-1 gene, can be used as a transgene.Alternatively, a B7-4 or PD-1 gene homologue, such as another B7-4 orPD-1 family member, can be isolated based on hybridization to the B7-4or PD-1 family cDNA sequences of SEQ ID NO:1, 3, 10, or 11 (describedfurther in subsection I above) and used as a transgene. Intronicsequences and polyadenylation signals can also be included in thetransgene to increase the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to a B7-4or PD-1 transgene to direct expression of a B7-4 or PD-1 protein toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B. Manipulating the MouseEmbryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of a B7-4 or PD-1 transgene in its genome and/or expression ofB7-4 or PD-1 mRNA in tissues or cells of the animals. A transgenicfounder animal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encoding aB7-4 or PD-1 protein can further be bred to other transgenic animalscarrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a B7-4 or PD-1 gene into which adeletion, addition or substitution has been introduced to thereby alter,e.g., functionally disrupt, the B7-4 or PD-1 gene. The B7-4 or PD-1 genecan be a human gene (e.g., the SEQ ID NO:1, 3, 10, or 11), but morepreferably, is a non-human homologue of a human B7-4 or PD-1 gene (e.g.,a cDNA isolated by stringent hybridization with the nucleotide sequenceof SEQ ID NO:1, 3, 10, or 11). For example, a mouse B7-4 or PD-1 genecan be used to construct a homologous recombination vector suitable foraltering an endogenous B7-4 or PD-1 gene in the mouse genome. In apreferred embodiment, the vector is designed such that, upon homologousrecombination, the endogenous B7-4 or PD-1 gene is functionallydisrupted (i.e., no longer encodes a functional protein; also referredto as a “knock out” vector). Alternatively, the vector can be designedsuch that, upon homologous recombination, the endogenous B7-4 or PD-1gene is mutated or otherwise altered but still encodes a functionalprotein (e.g., the upstream regulatory region can be altered to therebyalter the expression of the endogenous B7-4 or PD-1 protein). In thehomologous recombination vector, the altered portion of the B7-4 or PD-1gene is flanked at its 5′ and 3′ ends by additional nucleic acidsequence of the B7-4 or PD-1 gene to allow for homologous recombinationto occur between the exogenous B7-4 or PD-1 gene carried by the vectorand an endogenous B7-4 or PD-1 gene in an embryonic stem cell. Theadditional flanking B7-4 or PD-1 nucleic acid sequence is of sufficientlength for successful homologous recombination with the endogenous gene.Typically, several kilobases of flanking DNA (both at the 5′ and 3′ends) are included in the vector (see e.g., Thomas, K. R. and Capecchi,M. R. (1987) Cell 51:503 for a description of homologous recombinationvectors). The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced B7-4 orPD-1 gene has homologously recombined with the endogenous B7-4 or PD-1gene are selected (see, e.g., Li, E. et al. (1992) Cell 69:915). Theselected cells are then injected into a blastocyst of an animal (e.g., amouse) to form aggregation chimeras (see e.g., Bradley, A. inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach,Robertson, E. J., ed. (IRL, Oxford, 1987) pp. 113-152). A chimericembryo can then be implanted into a suitable pseudopregnant femalefoster animal and the embryo brought to term. Progeny harboring thehomologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination vectors and homologous recombinantanimals are described further in Bradley, A. (1991) Curr. Opin.Biotechnol. 2:823-829 and in PCT International Publication Nos.: WO90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et al.; WO92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et al.

In addition to the foregoing, the skilled artisan will appreciate thatother approaches known in the art for homologous recombination can beapplied to the instant invention. Enzyme-assisted site-specificintegration systems are known in the art and can be applied to integratea DNA molecule at a predetermined location in a second target DNAmolecule. Examples of such enzyme-assisted integration systems includethe Cre recombinase-lox target system (e.g., as described in Baubonis,W. and Sauer, B. (1993) Nucl. Acids Res. 21:2025-2029; and Fukushige, S.and Sauer, B. (1992) Proc. Natl. Acad. Sci. USA 89:7905-7909) and theFLP recombinase-FRT target system (e.g., as described in Dang, D. T. andPerrimon, N. (1992) Dev. Genet. 13:367-375; and Fiering, S. et al.(1993) Proc. Natl. Acad. Sci. USA 90:8469-8473). Tetracycline-regulatedinducible homologous recombination systems, such as described in PCTPublication No. WO 94/29442 and PCT Publication No. WO 96/01313, alsocan be used.

For example, in another embodiment, transgenic non-humans animals can beproduced which contain selected systems which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc.Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinasesystem is the FLP recombinase system of Saccharomyces cerevisiae(O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinasesystem is used to regulate expression of the transgene, animalscontaining transgenes encoding both the Cre recombinase and a selectedprotein are required. Such animals can be provided through theconstruction of “double” transgenic animals, e.g., by mating twotransgenic animals, one containing a transgene encoding a selectedprotein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(O) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyte and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

V. Pharmaceutical Compositions

B7-4 or PD-1 modulators (e.g., B7-4 or PD-1 inhibitory or stimulatoryagents, including B7-4 or PD-1 nucleic acid molecules, proteins,antibodies described above, or compounds identified as modulators of aB7-4 or PD-1 activity and/or expression or modulators of the interactionbetween B7-4 and PD-1) can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the nucleic acid molecule, protein, or antibody and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a B7-4 or PD-1 protein or anti-B7-4 or PD-1 antibody) inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, modulatory agents are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations should be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

VI. Uses and Methods of the Invention

The B7-4 and/or PD-1 modulatory agents, e.g., the nucleic acidmolecules, proteins, protein homologues, and antibodies describedherein, can be used in one or more of the following methods: a) methodsof treatment, e.g., by up- or down-modulating the immune response; b)screening assays; c) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics).The isolated nucleic acid molecules of the invention can be used, forexample, to express B7-4 or PD-1 protein (e.g., via a recombinantexpression vector in a host cell in gene therapy applications), todetect B7-4 or PD-1 mRNA (e.g., in a biological sample) or a geneticalteration in a B7-4 or PD-1 gene, and to modulate B7-4 or PD-1activity, as described further below. The B7-4 or PD-1 proteins can beused to treat disorders characterized by insufficient or excessiveproduction of B7-4 or PD-1 inhibitors. In addition, the B7-4 or PD-1proteins can be used to screen for naturally occurring B7-4 or PD-1binding partners, to screen for drugs or compounds which modulate B7-4or PD-1 activity, as well as to treat disorders characterized byinsufficient or excessive production of B7-4 or PD-1 protein orproduction of B7-4 or PD-1 protein forms which have decreased oraberrant activity compared to B7-4 or PD-1 wild type protein. Moreover,the anti-B7-4 or PD-1 antibodies of the invention can be used to detectand isolate B7-4 or PD-1 proteins, regulate the bioavailability of B7-4or PD-1 proteins, and modulate B7-4 or PD-1 activity e.g., by modulatingthe interaction of B7-4 and PD-1.

A. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant B7-4 or PD-1 expression oractivity.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant B7-4 or PD-1expression or activity, by administering to the subject a B7-4 or PD-1polypeptide or an agent which modulates B7-4 or PD-1 polypeptideexpression or at least one B7-4 or PD-1 activity. Subjects at risk for adisease which is caused or contributed to by aberrant B7-4 or PD-1expression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of B7-4 or PD-1 aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of B7-4 or PD-1 aberrancy orcondition, for example, a B7-4 or PD-1 polypeptide, B7-4 or PD-1 agonistor B7-4 or PD-1 antagonist agent can be used for treating the subject.The appropriate agent can be determined based on clinical indicationsand can be identified, e.g., using screening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating B7-4or PD-1 expression or activity for therapeutic purposes. B7-4 has beendemonstrated to inhibit the costimulation and proliferation of activatedimmune cells and to transmit an inhibitory signal to immune cells viaPD-1. Accordingly, the activity and/or expression of B7-4 or PD-1 aswell as the interaction between B7-4 and PD-1 can be modulated in orderto modulate the immune response. It should be understood that inembodiments where B7-4 binds to a costimulatory receptor, upregulationof B7-4 activity results in upregulation of immune responses, whereasdownregulation of B7-4 activity results in downregulation of immuneresponses. In embodiments where B7-4 binds to inhibitory receptors,upregulation of B7-4 activity results in downregulation of immuneresponses, whereas downregulation of B7-4 activity results inupregulation of immune responses. In a preferred embodiment, B7-4 bindsto inhibitory receptors. In a particularly preferred embodiment, B7-4binds to PD-1.

Modulatory methods of the invention involve contacting a cell with amodulator of a B7-4 or a PD-1 polypeptide, e.g., an agent that modulatesexpression or activity of B7-4 and/or PD-1, or an agent that modulatesthe interaction of B7-4 and PD-1.

An agent that modulates B7-4 or PD-1 protein activity is an agent asdescribed herein, such as a nucleic acid or a protein molecule, anaturally-occurring target molecule of a B7-4 or PD-1 protein (e.g.,PD-1 in the case of B7-4 or B7-4 in the case of PD-1), a B7-4 or PD-1antibody, a B7-4 or PD-1 agonist or antagonist, a peptidomimetic of aB7-4 or PD-1 agonist or antagonist, or other small molecule.

An agent that modulates the expression of B7-4 or PD-1 is, e.g., anantisense nucleic acid molecule, triplex oligonucleotide, or a ribozymeor a recombinant vector for expression of a B7-4 or PD-1 protein. Forexample, an oligonucleotide complementary to the area around a B7-4 orPD-1 polypeptide translation initiation site, can be synthesized. One ormore antisense oligonucleotides can be added to cell media, typically at200 μg/ml, or administered to a patient to prevent the synthesis of aB7-4 or PD-1 polypeptide. The antisense oligonucleotide is taken up bycells and hybridizes to a B7-4 or PD-1 mRNA to prevent translation.Alternatively, an oligonucleotide which binds double-stranded DNA toform a triplex construct to prevent DNA unwinding and transcription canbe used. As a result of either, synthesis of a B7-4 or PD-1 polypeptideis blocked. When PD-1 expression is modulated, preferably, suchmodulation occurs by a means other than by knocking out the PD-1 gene.

Agents which modulate expression, by virtue of the fact that theycontrol the amount of PD-1 or B7-4 in a cell, also modulate the totalamount of PD-1 or B7-4 activity in a cell.

In one embodiment, an agent that stimulates an inhibitory or activity ofB7-4 or an inhibitory activity of PD-1 is an agonist of B7-4 or PD-1.Examples of such agents include active B7-4 or PD-1 protein and anucleic acid molecule encoding B7-4 or PD-1 polypeptide that has beenintroduced into the cell.

In another embodiment, the agent inhibits the costimulatory orinhibitory activity of B7-4 or inhibitory activity of PD-1 and is anantagonist of B7-4 or PD-1. Examples of such agents include antisenseB7-4 or PD-1 nucleic acid molecules, anti-B7-4 or PD-1 antibodies,soluble, nonactivating forms of B7-4 or PD-1 molecules, and B7-4 or PD-1inhibitors.

These modulatory agents can be administered in vitro (e.g., bycontacting the cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder that would benefit from modulation of a B7-4 or PD-1 protein,e.g., a disorder which would benefit from up or downmodulation of theimmune response, or which is characterized by aberrant expression oractivity of a B7-4 or PD-1 protein or nucleic acid molecule. In oneembodiment, the method involves administering an agent (e.g., an agentidentified by a screening assay described herein), or combination ofagents that modulates (e.g., upregulates or downregulates) B7-4 or PD-1expression or activity. In another embodiment, the method involvesadministering a B7-4 or PD-1 protein or nucleic acid molecule as therapyto compensate for reduced or aberrant B7-4 or PD-1 expression oractivity.

Stimulation of B7-4 or PD-1 activity is desirable in situations in whichB7-4 or PD-1 is abnormally downregulated and/or in which increased B7-4or PD-1 activity is likely to have a beneficial effect. Likewise,inhibition of B7-4 or PD-1 activity is desirable in situations in whichB7-4 or PD-1 is abnormally upregulated and/or in which decreased B7-4 orPD-1 activity is likely to have a beneficial effect. One of ordinaryskill in the art should recognize that in embodiments where B7-4 bindsto a costimulatory, stimulation of B7-4 and stimulation of PD-1 haveopposite effects on immune cell costimulation, and therefore, on theimmune response. In such an instance, when stimulation of the activityof one molecule is desirable, suppression of the activity of the othermolecule is desirable.

Exemplary agents for use in downmodulating B7-4 (B7-4 antagonists)include (for example): antisense molecules, antibodies that recognizeB7-4, compounds that block interaction of B7-4 and one of its naturallyoccurring receptors on a immune cell (e.g., soluble, monovalent B7-4molecules, and soluble forms of B7-4 ligands or compounds identified inthe subject screening assays). Exemplary agents for use indownmodulating PD-1 (PD-1 antagonists) include (for example): antisensemolecules, antibodies that bind to PD-1, but do not transduce aninhibitory signal to the immune cell (“non-activating antibodies”), andsoluble forms of PD-1.

Exemplary agents for use in upmodulating B7-4 (B7-4 agonists) include(for example): nucleic acid molecules encoding B7-4 polypeptides,multivalent forms of B7-4, compounds that increase the expression ofB7-4, and cells that express B7-4, etc. Exemplary agents for use inupmodulating PD-1 (PD-1 agonists) include (for example): antibodies thattransmit an inhibitory signal via PD-1, compounds that enhance theexpression of PD-1, nucleic acid molecules encoding PD-1, and forms ofB7-4 that transduce a signal via PD-1.

3. Downregulation of Immune Responses by Modulation of B7-4 or PD-1

There are numerous embodiments of the invention for upregulating theinhibitory function or downregulating the costimulatory function of aB7-4 polypeptide to thereby downregulate immune responses.Downregulation can be in the form of inhibiting or blocking an immuneresponse already in progress or may involve preventing the induction ofan immune response.

The functions of activated immune cells can be inhibited bydown-regulating immune cell responses or by inducing specific anergy inimmune cells, or both.

For example, anti-B7-4 antibodies or B7-4 polypeptides (e.g., soluble,monomeric forms of a B7-4 polypeptide such as B7-4-Ig), and/or anti-B7-4antibodies that block the interaction of B7-4 with a costimulatoryreceptor can be used to inhibit a costimulatory signal and, thus,downmodulate the immune response.

In addition, in embodiments where B7-4 binds to an inhibitory receptor,forms of B7-4 that bind to the inhibitory receptor, e.g., multivalentB7-4 on a cell surface, can be used to downmodulate the immune response.

Likewise, the PD-1 pathway can also be stimulated by the use of an agentto thereby downmodulate the immune response. Inhibition of theinteraction of B7-4 with a stimulatory receptor on an immune cell (e.g.,by using a soluble form of PD-1 and/or CTLA4) or activation of PD-1(e.g., using an activating antibody which cross-links PD-1) may providenegative signals to immune cells.

In one embodiment of the invention, an activating antibody used tostimulate PD-1 activity is a bispecific antibody. For example, such anantibody can comprise a PD-1 binding site and another binding site whichtargets a cell surface receptor on an immune cell, e.g., on a T cell, aB cell, or a myeloid cell. In one embodiment, such an antibody, inaddition to comprising a PD-1 binding site can further comprise abinding site which binds to a molecule which is in proximity to anactivating or inhibitory receptor, e.g., B-cell antigen receptor, aT-cell antigen receptor, or an Fc receptor in order to target themolecule to a specific cell population. For example, a CD3 antigen, aT-cell receptor chain, LFA-1, CD2, CTLA-4, immunoglobulin, B cellreceptor, Ig alpha, Ig beta, CD22, or Fc receptor could be used. Suchantibodies (or other bispecific agents) are art recognized and can beproduced, e.g., as described herein. Selection of this second antigenfor the bispecific antibody provides flexibility in selection of cellpopulation to be targeted for inhibition.

In another embodiment, the co-ligation of PD-1 and an activating orinhibitory receptor on a cell can enhance the generation of a negativesignal via PD-1. Such co-ligation can be accomplished e.g., by use of abispecific agent, e.g., a bispecific antibody as described herein havingspecificity for both PD-1 and a molecule associated with a receptor. Inanother embodiment, the use of a multivalent form of an agent thattransmits a negative signal via PD-1 can be used to enhance thetransmission of a negative signal via PD-1, e.g., an agent presented ona bead or on a surface. In another embodiment, such a multivalent agentcan comprise two specificities to achieve co-ligation of PD-1 and areceptor or a receptor associated molecule (e.g., a bead comprising antiCD3 and B7-4).

Agents that block or inhibit interaction of B7-4 with a costimulatoryreceptor (e.g., soluble forms of B7-4 or blocking antibodies to B7-4) aswell as agents that promote a B7-4-mediated inhibitory signal oragonists of PD-1 which activate PD-1 (e.g., PD-1 activating antibodiesor PD-1 activating small molecules) can be identified by their abilityto inhibit immune cell proliferation and/or effector function or toinduce anergy when added to an in vitro assay. For example, cells can becultured in the presence of an agent that stimulates signal transductionvia an activating receptor. A number of art recognized readouts of cellactivation can be employed to measure, e.g., cell proliferation oreffector function (e.g., antibody production, cytokine production,phagocytosis) in the presence of the activating agent. The ability of atest agent to block this activation can be readily determined bymeasuring the ability of the agent to effect a decrease in proliferationor effector function being measured.

In one embodiment of the invention, tolerance is induced againstspecific antigens by co-administering an antigen with a PD-1 agonist.For example, tolerance can be induced to specific proteins. In oneembodiment, immune responses to allergens or foreign proteins to whichan immune response is undesirable can be inhibited. For example,patients that receive Factor VIII frequently generate antibodies againstthis clotting factor. Co-administration of an agent that blocks aB7-4-mediated costimulatory signal or an agent that stimulates a PD-1mediated inhibitory signal in combination with recombinant factor VIII(or by physically linked to Factor VIII, e.g., by cross-linking) canresult in downmodulation.

In one embodiment, fusion proteins comprising a B7-4 first peptide fusedto a second peptide having an activity of another B lymphocyte antigen(e.g., B7-1 or B7-2), can be used to block interaction of B7-4 with acostimulatory receptor on a immune cell to downmodulate immuneresponses. Alternatively, two separate peptides (for example, a B7-4polypeptide with B7-2 and/or B7-1), or a combination of blockingantibodies (e.g., antibodies against a B7-4 polypeptide with anti-B7-2and/or anti-B7-1 monoclonal antibodies) can be combined as a singlecomposition or administered separately (simultaneously or sequentially)to downregulate immune cell mediated immune responses in a subject.Furthermore, a therapeutically active amount of one, or more peptideshaving a B7-4 polypeptide activity, with B7-1 and/or B7-1 activity canbe used in conjunction with other downmodulating reagents to influenceimmune responses. Examples of other immunomodulating reagents includeantibodies that block a costimulatory signal, (e.g., against CD28,ICOS), antibodies that activate an inhibitory signal via CTLA4, and/orantibodies against other immune cell markers (e.g., against CD40,against CD40 ligand, or against cytokines), fusion proteins (e.g.,CTLA4-Fc, PD-1-Fc), and immunosuppressive drugs, (e.g., rapamycin,cyclosporine A or FK506).

The B7-4 and/or PD-1 peptides may also be useful in the construction oftherapeutic agents which block immune cell function by destruction ofcells. For example, portions of a B7-4 or PD-1 polypeptide can be linkedto a toxin to make a cytotoxic agent capable of triggering thedestruction of cells to which it binds.

For making cytotoxic agents, polypeptides of the invention may belinked, or operatively attached, to toxins using techniques that areknown in the art, e.g., crosslinking or via recombinant DNA techniques.The preparation of immunotoxins is, in general, well known in the art(see, e.g., U.S. Pat. Nos. 4,340,535, and EP 44167, both incorporatedherein by reference). Numerous types of disulfide-bond containinglinkers are known which can successfully be employed to conjugate thetoxin moiety with a polypeptide. In one embodiment, linkers that containa disulfide bond that is sterically “hindered” are to be preferred, dueto their greater stability in vivo, thus preventing release of the toxinmoiety prior to binding at the site of action.

A wide variety of toxins are known that may be conjugated topolypeptides or antibodies of the invention. Examples include: numeroususeful plant-, fungus- or even bacteria-derived toxins, which, by way ofexample, include various A chain toxins, particularly ricin A chain,ribosome inactivating proteins such as saporin or gelonin,.alpha.-sarcin, aspergillin, restrictocin, ribonucleases such asplacental ribonuclease, angiogenic, diphtheria toxin, and pseudomonasexotoxin, etc. A preferred toxin moiety for use in connection with theinvention is toxin A chain which has been treated to modify or removecarbohydrate residues, deglycosylated A chain. (U.S. Pat. No.5,776,427).

Infusion of one or a combination of such cytotoxic agents, (e.g., B7-4ricin (alone or in combination with B7-2-ricin or B7-1-ricin), into apatient may result in the death of immune cells, particularly in lightof the fact that activated immune cells that express higher amounts ofB7-4 ligands. For example, because PD-1 is induced on the surface ofactivated lymphocytes, an antibody against PD-1 can be used to targetthe depletion of these specific cells by Fc-R dependent mechanisms or byablation by conjugating a cytotoxic drug (e.g., ricin, saporin, orcalicheamicin) to the antibody. In one embodiment, the antibody toxincan be a bispecific antibody. Such bispecific antibodies are useful fortargeting a specific cell population, e.g., using a marker found only ona certain type of cell, e.g., a TCR, BCR, or FcR molecule.

Downregulating or preventing B7-4 polypeptide costimulatory functions oractivating a B7-4 or a PD-1 inhibitory function (e.g., by stimulation ofthe negative signaling function of PD-1) is useful to downmodulate theimmune response, e.g., in situations of tissue, skin and organtransplantation, in graft-versus-host disease (GVHD), or in autoimmunediseases such as systemic lupus erythematosus, and multiple sclerosis.For example, blockage of immune cell function results in reduced tissuedestruction in tissue transplantation. Typically, in tissue transplants,rejection of the transplant is initiated through its recognition asforeign by immune cells, followed by an immune reaction that destroysthe transplant. The administration of a molecule which inhibits orblocks interaction of a B7 molecule with a costimulatory receptor(s) onimmune cells (such as a soluble, monomeric form of a B7-4 or PD-1polypeptide) alone or in conjunction with another downmodulatory agentprior to or at the time of transplantation can inhibit the generation ofa costimulatory signal. Moreover, inhibition of B7-4 costimulatorysignals, or promotion of a B7-4 or PD-1 inhibitory signal may also besufficient to anergize the immune cells, thereby inducing tolerance in asubject. Induction of long-term tolerance by blocking a B7-4 mediatedcostimulatory signal may avoid the necessity of repeated administrationof these blocking reagents.

To achieve sufficient immunosuppression or tolerance in a subject, itmay also be desirable to block the costimulatory function of othermolecules. For example, it may be desirable to block the function ofB7-1 and B7-4, B7-2 and B7-4, or B7-1 and B7-4 by administering asoluble form of a combination of peptides having an activity of each ofthese antigens or blocking antibodies against these antigens (separatelyor together in a single composition) prior to or at the time oftransplantation. Alternatively, it may be desirable to promoteinhibitory activity of B7-4 or PD-1 and inhibit a costimulatory activityof B7-1 and/or B7-2. Other downmodulatory agents that can be used inconnection with the downmodulatory methods of the invention include, forexample, agents that transmit an inhibitory signal via CTLA4, solubleforms of CTLA4, antibodies that activate an inhibitory signal via CTLA4,blocking antibodies against other immune cell markers or soluble formsof other receptor ligand pairs (e.g., agents that disrupt theinteraction between CD40 and CD40 ligand (e.g., anti CD40 ligandantibodies)), antibodies against cytokines, or immunosuppressive drugs.In another embodiment, a combination of at least two different B7-4antibodies can be administered to achieve optimal blocking activity.

For example, blocking B7-4 polypeptide costimulation or activating aB7-4 or PD-1 inhibitory function can also be useful in treatingautoimmune disease. Many autoimmune disorders are the result ofinappropriate activation of immune cells that are reactive against selftissue and which promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive immune cells may reduce or eliminate disease symptoms.Administration of reagents which block costimulation of immune cells bydisrupting receptor:ligand interactions of B7 molecules withcostimulatory receptors is useful to inhibit immune cell activation andprevent production of autoantibodies or cytokines which may be involvedin the disease process. Additionally, agents that promote an inhibitoryfunction of B7-4 or PD-1 may induce antigen-specific tolerance ofautoreactive immune cells which could lead to long-term relief from thedisease. The efficacy of reagents in preventing or alleviatingautoimmune disorders can be determined using a number ofwell-characterized animal models of human autoimmune diseases. Examplesinclude murine experimental autoimmune encephalitis systemic lupuserythematosus in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmunecollagen arthritis, diabetes mellitus in NOD mice and BB rats, andmurine experimental myasthenia gravis (see Paul ed., FundamentalImmunology, Raven Press, New York, 1989, pp. 840-856).

Inhibition of immune cell activation is useful therapeutically in thetreatment of allergy and allergic reactions, e.g., by inhibiting IgEproduction. An agent that promotes a B7-4 or PD-1 inhibitory functioncan be administered to an allergic subject to inhibit immune cellmediated allergic responses in the subject. Activating a PD-1polypeptide may also be useful in treating allergies. Inhibition of B7-4costimulation of immune cells or stimulation of a B7-4 or PD-1inhibitory pathway can be accompanied by exposure to allergen inconjunction with appropriate MHC molecules. Allergic reactions can besystemic or local in nature, depending on the route of entry of theallergen and the pattern of deposition of IgE on mast cells orbasophils. Thus, inhibition of immune cell mediated allergic responseslocally or systemically by administration of an inhibitory form of anagent that inhibits the interaction of B7-4 with a costimulatoryreceptor or an agent that promotes an inhibitory function of B7-4 orPD-1.

Inhibition of immune cell activation through blockage of a B7-4costimulatory activity or stimulation of PD-1 inhibitory activity mayalso be important therapeutically in viral infections of immune cells.For example, in the acquired immune deficiency syndrome (AIDS), viralreplication is stimulated by immune cell activation. Blocking aB7-4/costimulatory receptor interaction or stimulation of B7-4 or PD-1inhibitory function may result in inhibition of viral replication andthereby ameliorate the course of AIDS.

Downregulation of an immune response via stimulation of B7-4 activity orB7-4 interaction with its natural binding partner(s), e.g., PD-1, mayalso be useful in promoting the maintenance of pregnancy. B7-4 isnormally highly expressed in placental trophoblasts, the layer of cellsthat forms the interface between mother and fetus and may play a role inpreventing maternal rejection of the fetus. Females at risk forspontaneous abortion (e.g., those identified by screening for B7-4activity, as described in the “Prognostic Assays” section, those whohave previously had a spontaneous abortion or those who have haddifficulty conceiving) because of immunologic rejection of the embryo orfetus can be treated with agents that stimulate the activity of B7-4 orits interaction with its natural binding partner(s), e.g., PD-1.

Downregulation of an immune response via stimulation of B7-4 activity orB7-4 interaction with its natural binding partner(s), e.g., PD-1, mayalso be useful in treating an autoimmune attack of autologous tissuesFor example, B7-4 is normally highly expressed in the heart and protectsthe heart from autoimmune attack. This is evidenced by the fact that theBalb/c PD-1 knockout mouse exhibits massive autoimmune attack on theheart with thrombosis. Thus, conditions that are caused or exacerbatedby autoimmune attack (e.g., in this example, heart disease, myocardialinfarction or atherosclerosis) may be ameliorated or improved byincreasing B7-4 activity or B7-4 biding to its natural binding partner,e.g., PD-1. It is therefore within the scope of the invention tomodulate conditions exacerbated by autoimmune attack, such as autoimmunedisorders (as well as conditions such as heart disease, myocardialinfarction, and atherosclerosis) by stimulating B7-4 activity or B7-4interaction with B7-4.

4. Upregulation of Immune Responses

Upregulation of B7-4 costimulatory activity or inhibit an inhibitoryactivity of PD-1 or B7-4 as a means of upregulating immune responses isalso useful in therapy. Upregulation of immune responses can be in theform of enhancing an existing immune response or eliciting an initialimmune response. For example, enhancing an immune response throughstimulating B7-4 costimulatory activity or inhibition of B7-4 or PD-1inhibitory activity is useful in cases of infections with microbes,e.g., bacteria, viruses, or parasites. For example, in one embodiment, aform of B7-4 that promotes a costimulatory signal in an immune cell(e.g., a B7-4 peptide in a multi-valent form (e.g., a solublemultivalent form or a form expressed on a cell surface)) or an agentthat inhibits the interaction of B7-4 with an inhibitory receptor or anagent that inhibits transduction of an inhibitory signal via PD-1, e.g.,a non-activating antibody against PD-1, is therapeutically useful insituations where upregulation of antibody and cell-mediated responses,resulting in more rapid or thorough clearance of virus, would bebeneficial. These would include viral skin diseases such as Herpes orshingles, in which case such an agent can be delivered topically to theskin. In addition, systemic viral diseases such as influenza, the commoncold, and encephalitis might be alleviated by the administration of suchagents systemically.

In certain instances, it may be desirable to further administer otheragents that upregulate immune responses, for example, forms other B7family members that transduce signals via costimulatory receptors, inorder further augment the immune response.

Alternatively, immune responses can be enhanced in an infected patientby removing immune cells from the patient, contacting immune cells invitro with a form of B7-4 that promotes a costimulatory signal in animmune cell or an agent that inhibits the interaction of B7-4 with aninhibitory receptor, or an agent that inhibits transduction of aninhibitory signal via PD-1, and reintroducing the in vitro stimulatedimmune cells into the patient. In another embodiment, a method ofenhancing immune responses involves isolating infected cells from apatient, e.g., virally infected cells, transfecting them with a nucleicacid molecule encoding a form of B7-4 that binds to a costimulatoryreceptor such that the cells express all or a portion of the B7-4molecule on their surface, and reintroducing the transfected cells intothe patient. The transfected cells are capable of delivering acostimulatory signal to, and thereby activate, immune cells in vivo.

Forms of B7-4 that promote a costimulatory signal in an immune cell, oran agent that inhibits the interaction of B7-4 with an inhibitoryreceptor, or an agent that inhibits transduction of an inhibitory signalvia PD-1 can be used prophylactically in vaccines against variouspolypeptides, e.g., polypeptides derived from pathogens. Immunityagainst a pathogen, e.g., a virus, can be induced by vaccinating with aviral protein along with a form of B7-4 that promotes a costimulatorysignal in an immune cell, or an agent that inhibits the interaction ofB7-4 with an inhibitory receptor, or an agent that inhibits transductionof an inhibitory signal via PD-1 in an appropriate adjuvant.Alternately, a vector comprising genes which encode for both apathogenic antigen and a form of B7-4 that binds to costimulatoryreceptors can be used for vaccination. Nucleic acid vaccines can beadministered by a variety of means, for example, by injection (e.g.,intramuscular, intradermal, or the biolistic injection of DNA-coatedgold particles into the epidermis with a gene gun that uses a particleaccelerator or a compressed gas to inject the particles into the skin(Haynes et al. 1996. J. Biotechnol. 44:37)). Alternatively, nucleic acidvaccines can be administered by non-invasive means. For example, pure orlipid-formulated DNA can be delivered to the respiratory system ortargeted elsewhere, e.g., Peyers patches by oral delivery of DNA(Schubbert. 1997. Proc. Natl. Acad. Sci. USA 94:961). Attenuatedmicroorganisms can be used for delivery to mucosal surfaces. (Sizemoreet al. 1995. Science. 270:29)

In one embodiment, a form of a B7-4 polypeptide which transmits acostimulatory signal can be administered with class I MHC proteins by,for example, a cell transfected to coexpress a B7-4 polypeptide and MHCclass I α chain protein and β₂ microglobulin to result in activation ofT cells and provide immunity from infection. For example, pathogens forwhich vaccines are useful include hepatitis B, hepatitis C, Epstein-Barrvirus, cytomegalovirus, HIV-1, HIV-2, tuberculosis, malaria andschistosomiasis.

In another application, upregulation or enhancement of a B7-4costimulatory function is useful in the induction of tumor immunity.Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma,carcinoma) transfected with a nucleic acid molecule encoding a B7-4antigen can be administered to a subject to overcome tumor-specifictolerance in the subject. If desired, the tumor cell can be transfectedto express a combination of B7 polypeptides (e.g., B7-1, B7-2, B7-4).For example, tumor cells obtained from a patient can be transfected exvivo with an expression vector directing the expression of a B7-4polypeptide alone, or in conjunction with a peptide having B7-1 activityand/or B7-2 activity. The transfected tumor cells are returned to thepatient to result in expression of the peptides on the surface of thetransfected cell. Alternatively, gene therapy techniques can be used totarget a tumor cell for transfection in vivo.

In addition, tumor cells which lack MHC class I or MHC class IImolecules, or which fail to express sufficient amounts of MHC class I orMHC class II molecules, can be transfected with nucleic acid encodingall or a portion of (e.g., a cytoplasmic-domain truncated portion) of anMHC class I α chain protein and β₂ microglobulin protein or an MHC classII α chain protein and an MHC class II β chain protein to therebyexpress MHC class I or MHC class II proteins on the cell surface.Expression of the appropriate class I or class II MHC in conjunctionwith a peptide having the activity of a B lymphocyte antigen (e.g.,B7-1, B7-2, B7-4) induces a T cell mediated immune response against thetransfected tumor cell. Optionally, a gene encoding an antisenseconstruct which blocks expression of an MHC class II associated protein,such as the invariant chain, can also be cotransfected with a DNAencoding a B7-4 polypeptide to promote presentation of tumor associatedantigens and induce tumor specific immunity. Expression of B7-1 by B7negative murine tumor cells has been shown to induce T cell mediatedspecific immunity accompanied by tumor rejection and prolongedprotection to tumor challenge in mice (Chen, L., et al. (1992) Cell 71,1093-1102; Townsend, S. E. and Allison, J. P. (1993) Science 259,368-370; Baskar, S., et al. (1993) Proc. Natl. Acad. Sci. 90,5687-5690). Thus, the induction of a immune cell mediated immuneresponse in a human subject can be sufficient to overcome tumor-specifictolerance in the subject.

In another embodiment, the immune response can be stimulated by thetransmission of a signal via a costimulatory receptor that binds to B7-4or by the inhibiting signaling via an inhibitory receptor that binds toB7-4, e.g.,PD-1, such that preexisting tolerance is overcome. Forexample, immune responses against antigens to which a subject cannotmount a significant immune response, e.g., to an autologous antigen,such as a tumor specific antigens can be induced by administering anagent that inhibits the inhibitory activity of PD-1 or the ability ofB7-4 to bind to an inhibitory ligand. For example, in one embodiment,soluble PD-1 or soluble B7-4 can be used (e.g., PD-1 Fc or B7-4 Fc) toenhance an immune response, e.g., to a tumor cell. In one embodiment, anautologous antigen, such as a tumor-specific antigen can becoadministered with an agent that inhibits the inhibitory activity ofPD-1 or the ability of B7-4 to bind to an inhibitory ligand. In anotherembodiment, an immune response can be stimulated against an antigen(e.g., an autologous antigen) to treat a neurological disorder. Inanother embodiment, PD-1 antagonists can be used as adjuvants to boostresponses to foreign antigens in the process of active immunization.

In yet another embodiment, the production of a form of B7-4 that bindsto an inhibitory receptor or that competes with the binding of B7-4 to acostimulatory receptor (e.g., a form of B7-4 that binds to PD-1 or anaturally occurring soluble molecule) can be inhibited, e.g., usingantisense RNA, in order to upregulate the immune response. For example,in one embodiment, the production of inhibitory B7-4 molecules by atumor cell can be inhibited in order to increase anti-tumor immunity.

In one embodiment, immune cells are obtained from a subject and culturedex vivo to in the presence of a form of B7-4 that binds a costimulatorymolecule or in the presence of an agent that that inhibits a B7-4 orPD-1 inhibitory signal, to expand the population of immune cells. In afurther embodiment the immune cells are then administered to a subject.Immune cells can be stimulated to proliferate in vitro by, for example,providing to the immune cells a primary activation signal and acostimulatory signal, as is known in the art. Various forms of B7-4proteins or agents that bind a costimulatory receptor or that inhibitsignaling via PD-1 can also be used to costimulate proliferation ofimmune cells. In one embodiment immune cells are cultured ex vivoaccording to the method described in PCT Application No. WO 94/29436.The costimulatory molecule can be soluble, attached to a cell membraneor attached to a solid surface, such as a bead.

B. Identification of Cytokines Modulated by Modulation of B7-4 and/orPD-1

The B7-4 and PD-1 molecules described herein can be used to identifycytokines which are produced by or whose production is enhanced orinhibited in immune cells in response to modulation of B7-4 and/or PD-1activity. Immune cells expressing PD-1 can be suboptimally stimulated invitro with a primary activation signal, for example, T cells can bestimulated with phorbol ester, anti-CD3 antibody or preferably antigenin association with an MHC class 11 molecule, and given a costimulatorysignal, e.g., by a stimulatory form of B7 family antigen, for instanceby a cell transfected with nucleic acid encoding a B7 polypeptide andexpressing the peptide on its surface or by a soluble, stimulatory formof the peptide. Known cytokines released into the media can beidentified by ELISA or by the ability of an antibody which blocks thecytokine to inhibit immune cell proliferation or proliferation of othercell types that is induced by the cytokine. For example, an IL-4 ELISAkit is available from Genzyme (Cambridge, Mass.), as is an IL-7 blockingantibody. Blocking antibodies against IL-9 and IL-12 are available fromGenetics Institute (Cambridge, Mass.). The effect of stimulating orblocking the interaction of B7-4 with PD-1 on the cytokine profile canthen be determined.

An in vitro immune cell costimulation assay as described above can alsobe used in a method for identifying novel cytokines which can bemodulated by modulation of B7-4 and or PD-1. For example, wherestimulation of the CD28/CTLA4 pathway seems to enhance IL-2 secretion,stimulation of the ICOS pathway seems to enhance IL-10 secretion(Hutloff et al. 199. Nature 397:263). If a particular activity inducedupon costimulation, e.g., immune cell proliferation, cannot be inhibitedby addition of blocking antibodies to known cytokines, the activity mayresult from the action of an unknown cytokine. Following costimulation,this cytokine can be purified from the media by conventional methods andits activity measured by its ability to induce immune cellproliferation.

To identify cytokines which may play a role in the induction oftolerance, an in vitro T cell costimulation assay as described above canbe used. In this case, T cells would be given the primary activationsignal and contacted with a selected cytokine, but would not be giventhe costimulatory signal. After washing and resting the immune cells,the cells would be rechallenged with both a primary activation signaland a costimulatory signal. If the immune cells do not respond (e.g.,proliferate or produce cytokines) they have become tolerized and thecytokine has not prevented the induction of tolerance. However, if theimmune cells respond, induction of tolerance has been prevented by thecytokine. Those cytokines which are capable of preventing the inductionof tolerance can be targeted for blockage in vivo in conjunction withreagents which block B lymphocyte antigens as a more efficient means toinduce tolerance in transplant recipients or subjects with autoimmunediseases. For example, one could administer a cytokine blocking antibodyto a subject along with an agent that promotes a B7-4 or a PD-1inhibitory activity.

C. Identification of Molecules which Modulate Expression of a B7-4 orPD-1 Polypeptide

The antibodies produced using the proteins and peptides of the currentinvention can be used in a screening assay for molecules which modulatethe expression of B7-4 or PD-1 polypeptide on cells. For example,molecules which modulate intracellular signaling pathways that culminatein changes in expression of B7-4 or PD-1 polypeptides (e.g., in responseto activation signals), can be identified by assaying expression of oneor more B7-4 or PD-1 polypeptides on the cell surface. Reducedimmunofluorescent staining by an appropriate antibody in the presence ofthe molecule would indicate that the molecule inhibits intracellularsignals. Molecules which upregulate B7-4 or PD-1 polypeptide expressionresult in an increased immunofluorescent staining. Alternatively, theeffect of a molecule on expression of a polypeptide can be determined bydetecting cellular mRNA levels using a probe of the invention. Forexample, a cell which expresses a B7-4 or PD-1 polypeptide can becontacted with a molecule to be tested, and an increase or decrease inmRNA levels in the cell detected by standard techniques, such asNorthern hybridization analysis or conventional dot blot of mRNA ortotal poly(A⁺)RNAs using a cDNA probe labeled with a detectable marker.Molecules which modulate expression of a B7-4 or PD-1 polypeptide areuseful therapeutically for either upregulating or downregulating immuneresponses alone or in conjunction with soluble blocking or stimulatingreagents as described above. For instance, a molecule which inhibitsexpression of B7-4 can be administered together with a second agent,e.g., an immunosuppressant or a molecule which inhibits expression ofPD-1 can be given with an immunostimulant, e.g., an adjuvant. Exemplarymolecules which can be tested for their ability to modulate B7-4 or PD-1include cytokines such as IL-4, γINF, IL-10, IL-12, GM-CSF andprostagladins.

D. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to B7-4 or PD-1 proteins, have a stimulatory or inhibitoryeffect on, for example, B7-4 or PD-1 expression or B7-4 or PD-1activity.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of a B7-4 orPD-1 protein or polypeptide or biologically active portion thereof,e.g., modulate the ability of B7-4 or PD-1 polypeptide to interact withits cognate binding partner or an interactor molecule (e.g., anintracellular interactor molecule). The test compounds of the presentinvention can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the‘one-bead one-compound’ library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andin Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds can be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner USP No. '409), plasmids (Cullet al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a B7-4 target molecule (an intracellularinteractor molecule or a PD-1 receptor) or PD-1 target molecule (e.g., aB7-4 ligand or intracellular interactor molecule) with a test compoundand determining the ability of the test compound to modulate (e.g.stimulate or inhibit) the activity of the B7-4 or PD-1 target molecule.Determining the ability of the test compound to modulate the activity ofa B7-4 or PD-1 target molecule can be accomplished, for example, bydetermining the ability of the B7-4 or PD-1 protein to bind to orinteract with the B7-4 or PD-1 target molecule. Determining the abilityof the B7-4 or PD-1 protein to bind to or interact with its bindingpartner can be accomplished, e.g., by measuring direct binding.

In a direct binding assay, the B7-4 or PD-1 protein (or their respectivetarget molecules) can be coupled with a radioisotope or enzymatic labelsuch that binding of the B7-4 or PD-1 protein to a B7-4 or PD-1 targetmolecule can be determined by detecting the labeled protein in acomplex. For example, B7-4 or PD-1 molecules, e.g., B7-4 or PD-1proteins, can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly orindirectly, and the radioisotope detected by direct counting ofradioemmission or by scintillation counting. Alternatively, B7-4 or PD-1molecules can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

It is also within the scope of this invention to determine the abilityof a compound to modulate the interaction between B7-4 or PD-1 and itstarget molecule, without the labeling of any of the interactants. Forexample, a microphysiometer can be used to detect the interaction ofB7-4 or PD-1 with its target molecule without the labeling of eitherB7-4 or PD-1 or the target molecule (McConnell, H. M. et al. (1992)Science 257:1906-1912). As used herein, a “microphysiometer” (e.g.,Cytosensor) is an analytical instrument that measures the rate at whicha cell acidifies its environment using a light-addressablepotentiometric sensor (LAPS). Changes in this acidification rate can beused as an indicator of the interaction between compound and receptor.

In a preferred embodiment, determining the ability of the B7-4 or PD-1protein to bind to or interact with a B7-4 or PD-1 target molecule canbe accomplished by determining the activity of the B7-4, PD-1 or theappropriate target molecule. For example, the activity of B7-4, PD-1 orthe appropriate target molecule can be determined by detecting inductionof a cellular second messenger (e.g., tyrosine kinase activity),detecting catalytic/enzymatic activity of an appropriate substrate,detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., chloramphenicol acetyltransferase), or detecting a cellular response regulated by B7-4, PD-1or the appropriate target molecule. For example, determining the abilityof the B7-4 or PD-1 protein to bind to or interact with a B7-4 or PD-1target molecule can be accomplished, for example, by measuring theability of a compound to modulate immune cell costimulation orinhibition in a proliferation assay, or by interfering with the abilityof a B7-4 or PD-1 polypeptide to bind to antibodies that recognize aportion of the B7-4 or PD-1 polypeptide.

In yet another embodiment, an assay of the present invention is acell-free assay in which a B7-4 or PD-1 protein or biologically activeportion thereof is contacted with a test compound and the ability of thetest compound to bind to the B7-4 or PD-1 protein or biologically activeportion thereof is determined. Binding of the test compound to the B7-4or PD-1 protein can be determined either directly or indirectly asdescribed above. In a preferred embodiment, the assay includescontacting the B7-4 or PD-1 protein or biologically active portionthereof with a known compound which binds B7-4 or PD-1 to form an assaymixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with a B7-4 orPD-1 protein, wherein determining the ability of the test compound tointeract with a B7-4 or PD-1 protein comprises determining the abilityof the test compound to preferentially bind to B7-4 or PD-1 polypeptideor biologically active portion thereof as compared to the knowncompound.

In another embodiment, the assay is a cell-free assay in which a B7-4 orPD-1 protein or biologically active portion thereof is contacted with atest compound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the B7-4 or PD-1 protein orbiologically active portion thereof is determined. Determining theability of the test compound to modulate the activity of a B7-4 or PD-1protein can be accomplished, for example, by determining the ability ofthe B7-4 or PD-1 protein to bind to a B7-4 or PD-1 target molecule byone of the methods described above for determining direct binding.Determining the ability of the B7-4 or PD-1 protein to bind to a B7-4 orPD-1 target molecule can also be accomplished using a technology such asreal-time Biomolecular Interaction Analysis (BIA) (Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” is atechnology for studying biospecific interactions in real time, withoutlabeling any of the interactants (e.g., BIAcore). Changes in the opticalphenomenon of surface plasmon resonance (SPR) can be used as anindication of real-time reactions between biological molecules.

In yet another embodiment, the cell-free assay involves contacting aB7-4 or PD-1 protein or biologically active portion thereof with a knowncompound which binds the B7-4 or PD-1 protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the B7-4 or PD-1 protein,wherein determining the ability of the test compound to interact withthe B7-4 or PD-1 protein comprises determining the ability of the B7-4or PD-1 protein to preferentially bind to or modulate the activity of aB7-4 or PD-1 target molecule.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of proteins (e.g., B7-4 or PD-1proteins or biologically active portions thereof, or binding partners towhich B7-4 or PD-1 binds). In the case of cell-free assays in which amembrane-bound form a protein is used (e.g., a cell surface B7-4 or PD-1receptor) it may be desirable to utilize a solubilizing agent such thatthe membrane-bound form of the protein is maintained in solution.Examples of such solubilizing agents include non-ionic detergents suchas n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton™ X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either B7-4or PD-1 or anappropriate target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to aB7-4 or PD-1 protein, or interaction of a B7-4 or PD-1 protein with atarget molecule in the presence and absence of a candidate compound, canbe accomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase/B7-4 orPD-1 fusion proteins or glutathione-S-transferase/target fusion proteinscan be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.Louis, Mo.) or glutathione derivatized microtiter plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target protein or B7-4 or PD-1 protein, and the mixtureincubated under conditions conducive to complex formation (e.g., atphysiological conditions for salt and pH). Following incubation, thebeads or microtiter plate wells are washed to remove any unboundcomponents, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as describedabove. Alternatively, the complexes can be dissociated from the matrix,and the level of B7-4 or PD-1 binding or activity determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a B7-4 orPD-1 protein or a B7-4 or PD-1 target molecule can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylated B7-4 orPD-1 protein or target molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with B7-4 or PD-1 protein or targetmolecules but which do not interfere with binding of the B7-4 or PD-1protein to its target molecule can be derivatized to the wells of theplate, and unbound target or B7-4 or PD-1 protein trapped in the wellsby antibody conjugation. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theB7-4 or PD-1 protein or target molecule, as well as enzyme-linked assayswhich rely on detecting an enzymatic activity associated with the B7-4or PD-1 protein or target molecule.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a B7-4 or PD-1 protein can beaccomplished by determining the ability of the test compound to modulatethe activity of a molecule that functions downstream of B7-4, e.g., amolecule that interacts with B7-4, or a molecule that functionsdownstream of PD-1, e.g., by interacting with the cytoplasmic domain ofPD-1. For example, levels of second messengers can be determined, theactivity of the interactor molecule on an appropriate target can bedetermined, or the binding of the interactor to an appropriate targetcan be determined as previously described.

In another embodiment, modulators of B7-4 or PD-1 expression areidentified in a method wherein a cell is contacted with a candidatecompound and the expression of B7-4 or PD-1 mRNA or protein in the cellis determined. The level of expression of B7-4 or PD-1 mRNA or proteinin the presence of the candidate compound is compared to the level ofexpression of B7-4 or PD-1 mRNA or protein in the absence of thecandidate compound. The candidate compound can then be identified as amodulator of B7-4 or PD-1 expression based on this comparison. Forexample, when expression of B7-4 or PD-1 mRNA or protein is greater(e.g., statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of B7-4 or PD-1 mRNA or protein expression.Alternatively, when expression of B7-4 or PD-1 mRNA or protein is less(e.g., statistically significantly less) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as an inhibitor of B7-4 or PD-1 mRNA or protein expression.The level of B7-4 or PD-1 mRNA or protein expression in the cells can bedetermined by methods described herein for detecting B7-4 or PD-1 mRNAor protein.

In yet another aspect of the invention, the B7-4 or PD-1 proteins,preferably in membrane bound form, can be used as “bait proteins” in atwo-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J.Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and BrentWO94/10300), to identify other proteins (“B7-4or PD-1 binding proteins”or “B7-4 or PD-1 bp”), which bind to or interact with B7-4 or PD-1 andare involved in B7-4 or PD-1 activity. Such B7-4-or PD-1 bindingproteins are also likely to be involved in the propagation of signals bythe B7-4 or PD-1 proteins or B7-4 or PD-1 targets as, for example,upstream or downstream elements of a B7-4 or PD-1 mediated signalingpathway. Alternatively, such B7-4 or PD-1 binding proteins may be B7-4or PD-1 inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a B7-4 or PD-1protein is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aB7-4-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the B7-4 or PD-1 protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a B7-4 or PD-1 modulating agent, an antisenseB7-4 or PD-1 nucleic acid molecule, a B7-4-or PD-1 specific antibody, ora B7-4 or PD-1 binding partner) can be used in an animal model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

F. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of B7-4 nucleotide sequences, described herein,can be used to map the location of B7-4 genes on a chromosome. Themapping of B7-4 sequences to chromosomes is an important first step incorrelating these sequences with genes associated with disease.

Briefly, B7-4 genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from B7-4 nucleotide sequences.Computer analysis of B7-4 sequences can be used to predict primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers can then be used for PCR screeningof somatic cell hybrids containing individual human chromosomes. Onlythose hybrids containing the human gene corresponding to B7-4 sequenceswill yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media, panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes. (D'Eustachio, P. et al. (1983)Science 220:919-924). Somatic cell hybrids containing only fragments ofhuman chromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using B7-4nucleotide sequences to design oligonucleotide primers, sublocalizationcan be achieved with panels of fragments from specific chromosomes.Other mapping strategies which can similarly be used to map a sequenceto its chromosome include in situ hybridization (described in Fan, Y. etal. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening withlabeled flow-sorted chromosomes, and pre-selection by hybridization tochromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in McKusick,V., Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the B7-4 gene can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The B7-4 sequences of the present invention can also be used to identifyindividuals from minute biological samples. The United States military,for example, is considering the use of restriction fragment lengthpolymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the B7-4 nucleotide sequences described herein can be usedto prepare two PCR primers from the 5′ and 3′ ends of the sequences.These primers can then be used to amplify an individual's DNA andsubsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The B7-4 nucleotide sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO:1 or 3can comfortably provide positive individual identification with a panelof perhaps 10 to 1,000 primers which each yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences are used, a moreappropriate number of primers for positive individual identificationwould be 500-2,000.

If a panel of reagents from B7-4 nucleotide sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

3. Use of Partial B7-4 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions are particularly appropriate for this useas greater numbers of polymorphisms occur in the noncoding regions,making it easier to differentiate individuals using this technique.Examples of polynucleotide reagents include the B7-4 nucleotidesequences or portions thereof having a length of at least 20 bases,preferably at least 30 bases.

The B7-4 nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., brain tissue. This can be very usefulin cases where a forensic pathologist is presented with a tissue ofunknown origin. Panels of such B7-4 probes can be used to identifytissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., B7-4 primers or probes canbe used to screen tissue culture for contamination (i.e. screen for thepresence of a mixture of different types of cells in a culture).

G. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining B7-4 or PD-1protein and/or nucleic acid expression as well as B7-4 or PD-1 activity,in the context of a biological sample (e.g., blood, serum, cells,tissue) to thereby determine whether an individual is afflicted with adisease or disorder, or is at risk of developing a disorder, associatedwith aberrant B7-4 or PD-1 expression or activity. The invention alsoprovides for prognostic (or predictive) assays for determining whetheran individual is at risk of developing a disorder associated with B7-4or PD-1 protein, nucleic acid expression or activity. For example,mutations in a B7-4 or PD-1 gene can be assayed in a biological sample.Such assays can be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with B7-4 or PD-1 protein, nucleic acidexpression or activity. The assays described herein, such as thepreceding diagnostic assays or the following assays, can also be used todetect a tendency to have spontaneous abortions.

Another aspect of the invention pertains to monitoring the influence ofagents (e.g., drugs, compounds) on the expression or activity of B7-4 orPD-1 in clinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of B7-4 orPD-1 protein or nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting B7-4 or PD-1protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes B7-4 orPD-1 protein such that the presence of B7-4 or PD-1 protein or nucleicacid is detected in the biological sample. A preferred agent fordetecting B7-4 or PD-1 mRNA or genomic DNA is a labeled nucleic acidprobe capable of hybridizing to B7-4 or PD-1 mRNA or genomic DNA. Thenucleic acid probe can be, for example, a human B7-4 or PD-1 nucleicacid, such as the nucleic acid of SEQ ID NO:1, 3, 10, or 11 or a portionthereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or500 nucleotides in length and sufficient to specifically hybridize understringent conditions to B7-4 or PD-1 mRNA or genomic DNA. Other suitableprobes for use in the diagnostic assays of the invention are describedherein.

A preferred agent for detecting B7-4 or PD-1 protein is an antibodycapable of binding to B7-4 or PD-1 protein, preferably an antibody witha detectable label. Antibodies can be polyclonal, or more preferably,monoclonal. An intact antibody, or a fragment thereof (e.g., Fab orF(ab′)₂) can be used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin. The term “biological sample” is intended toinclude tissues, cells and biological fluids isolated from a subject, aswell as tissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect B7-4 or PD-1mRNA, protein, or genomic DNA in a biological sample in vitro as well asin vivo. For example, in vitro techniques for detection of B7-4 or PD-1mRNA include Northern hybridizations and in situ hybridizations. Invitro techniques for detection of B7-4 or PD-1 protein include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of B7-4 or PD-1 genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of B7-4 or PD-1 proteininclude introducing into a subject a labeled anti-B7-4 or PD-1 antibody.For example, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a serum sample isolated byconventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting B7-4 or PD-1 protein,mRNA, or genomic DNA, such that the presence of B7-4 or PD-1 protein,mRNA or genomic DNA is detected in the biological sample, and comparingthe presence of B7-4 or PD-1 protein, mRNA or genomic DNA in the controlsample with the presence of B7-4 or PD-1 protein, mRNA or genomic DNA inthe test sample.

The invention also encompasses kits for detecting the presence of B7-4or PD-1 in a biological sample. For example, the kit can comprise alabeled compound or agent capable of detecting B7-4 or PD-1 protein ormRNA in a biological sample; means for determining the amount of B7-4 orPD-1 in the sample; and means for comparing the amount of B7-4 or PD-1in the sample with a standard. The compound or agent can be packaged ina suitable container. The kit can further comprise instructions forusing the kit to detect B7-4 or PD-1 protein or nucleic acid.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant B7-4 or PD-1 expression or activity. Forexample, the assays described herein, such as the preceding diagnosticassays or the following assays, can be utilized to identify a subjecthaving or at risk of developing a disorder associated with B7-4 or PD-1protein, expression or activity. Thus, the present invention provides amethod for identifying a disease or disorder associated with aberrantB7-4 or PD-1 expression or activity in which a test sample is obtainedfrom a subject and B7-4 or PD-1 protein or nucleic acid (e.g., mRNA,genomic DNA) is detected, wherein the presence of B7-4 or PD-1 proteinor nucleic acid is diagnostic for a subject having or at risk ofdeveloping a disease or disorder associated with aberrant B7-4 or PD-1expression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., serum), cell sample, ortissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant B7-4 or PD-1 expression or activity. Thus, thepresent invention provides methods for determining whether a subject canbe effectively treated with an agent for a disorder associated withaberrant B7-4 or PD-1 expression or activity in which a test sample isobtained and B7-4 or PD-1 protein or nucleic acid expression or activityis detected (e.g., wherein the abundance of B7-4 or PD-1 protein ornucleic acid expression or activity is diagnostic for a subject that canbe administered the agent to treat a disorder associated with aberrantB7-4 or PD-1 expression or activity).

The methods of the invention can also be used to detect geneticalterations in a B7-4 or PD-1 gene, thereby determining if a subjectwith the altered gene is at risk for a disorder associated with the B7-4or PD-1 gene. In preferred embodiments, the methods include detecting,in a sample of cells from the subject, the presence or absence of agenetic alteration characterized by at least one of an alterationaffecting the integrity of a gene encoding a B7-4 or PD-1 protein, orthe mis-expression of the B7-4 or PD-1 gene. For example, such geneticalterations can be detected by ascertaining the existence of at leastone of 1) a deletion of one or more nucleotides from a B7-4 or PD-1gene; 2) an addition of one or more nucleotides to a B7-4 or PD-1 gene;3) a substitution of one or more nucleotides of a B7-4 or PD-1 gene, 4)a chromosomal rearrangement of a B7-4 or PD-1 gene; 5) an alteration inthe level of a messenger RNA transcript of a B7-4 or PD-1 gene, 6)aberrant modification of a B7-4 or PD-1 gene, such as of the methylationpattern of the genomic DNA, 7) the presence of a non-wild type splicingpattern of a messenger RNA transcript of a B7-4 or PD-1 gene, 8) anon-wild type level of a B7-4 or PD-1 protein, 9) allelic loss of a B7-4or PD-1 gene, and 10) inappropriate post-translational modification of aB7-4 or PD-1 protein. As described herein, there are a large number ofassay techniques known in the art which can be used for detectingalterations in a B7-4 or PD-1 gene. A preferred biological sample is atissue or serum sample isolated by conventional means from a subject,e.g., a cardiac tissue sample.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the B7-4 or PD-1gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). Thismethod can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a B7-4 or PD-1 gene underconditions such that hybridization and amplification of the B7-4 or PD-1gene (if present) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Biotechnology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a B7-4 or PD-1 gene from asample cell can be identified by alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in B7-4 or PD-1 can beidentified by hybridizing a sample and control nucleic acids, e.g., DNAor RNA, to high density arrays containing hundreds or thousands ofoligonucleotides probes (Cronin, M. T. et al. (1996) Hum. Mutat.7:244-255; Kozal, M. J. et al. (1996) Nat. Med. 2:753-759). For example,genetic mutations in B7-4 or PD-1 can be identified in two dimensionalarrays containing light-generated DNA probes as described in Cronin, M.T. et al. (1996) supra. Briefly, a first hybridization array of probescan be used to scan through long stretches of DNA in a sample andcontrol to identify base changes between the sequences by making lineararrays of sequential overlapping probes. This step allows theidentification of point mutations. This step is followed by a secondhybridization array that allows the characterization of specificmutations by using smaller, specialized probe arrays complementary toall variants or mutations detected. Each mutation array is composed ofparallel probe sets, one complementary to the wild-type gene and theother complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the B7-4 or PD-1 geneand detect mutations by comparing the sequence of the sample B7-4 orPD-1 with the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger((1977) Proc. Natl. Acad Sci. USA 74:5463). It is also contemplated thatany of a variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

Other methods for detecting mutations in the B7-4 or PD-1 gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.(1985) Science 230:1242). In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes formed by hybridizing(labeled) RNA or DNA containing the wild-type B7-4 or PD-1 sequence withpotentially mutant RNA or DNA obtained from a tissue sample. Thedouble-stranded duplexes are treated with an agent which cleavessingle-stranded regions of the duplex such as which will exist due tobasepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with SI nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al.(1992) Methods Enzymol. 217:286-295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in B7-4 or PD-1 cDNAs obtainedfrom samples of cells. For example, the mutY enzyme of E. coli cleaves Aat G/A mismatches and the thymidine DNA glycosylase from HeLa cellscleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662). According to an exemplary embodiment, a probe based on aB7-4 sequence, e.g., a wild-type B7-4 or PD-1 sequence, is hybridized toa cDNA or other DNA product from a test cell(s). The duplex is treatedwith a DNA mismatch repair enzyme, and the cleavage products, if any,can be detected from electrophoresis protocols or the like. See, forexample, U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility can beused to identify mutations in B7-4 or PD-1 genes. For example, singlestrand conformation polymorphism (SSCP) can be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci. USA: 86:2766,see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992)Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments ofsample and control B7-4 or PD-1 nucleic acids can be denatured andallowed to renature. The secondary structure of single-stranded nucleicacids varies according to sequence, the resulting alteration inelectrophoretic mobility enables the detection of even a single basechange. The DNA fragments can be labeled or detected with labeledprobes. The sensitivity of the assay may be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In a preferred embodiment, the subject methodutilizes heteroduplex analysis to separate double stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAcan be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification can be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner et al. (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein can be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which can be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a B7-4 or PD-1 gene.

Furthermore, any cell type or tissue in which B7-4 or PD-1 is expressedcan be utilized in the prognostic assays described herein.

VII. Administration of B7-4 or PD-1 Modulating Agents

B7-4 or PD-1 modulating agents of the invention are administered tosubjects in a biologically compatible form suitable for pharmaceuticaladministration in vivo to either enhance or suppress immune cellmediated immune responses. By “biologically compatible form suitable foradministration in vivo” is meant a form of the protein to beadministered in which any toxic effects are outweighed by thetherapeutic effects of the protein. The term subject is intended toinclude living organisms in which an immune response can be elicited,e.g., mammals. Examples of subjects include humans, dogs, cats, mice,rats, and transgenic species thereof. Administration of an agent asdescribed herein can be in any pharmacological form including atherapeutically active amount of an agent alone or in combination with apharmaceutically acceptable carrier.

Administration of a therapeutically active amount of the therapeuticcompositions of the present invention is defined as an amount effective,at dosages and for periods of time necessary to achieve the desiredresult. For example, a therapeutically active amount of a B7-4 or PD-1polypeptide may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of peptide toelicit a desired response in the individual. Dosage regimens can beadjusted to provide the optimum therapeutic response. For example,several divided doses can be administered daily or the dose can beproportionally reduced as indicated by the exigencies of the therapeuticsituation.

The B7-4 or PD-1 modulating agent (e.g., a peptide, a nucleic acidmolecule, antibody, peptidomimetic, or small molecule) can beadministered in a convenient manner such as by injection (subcutaneous,intravenous, etc.), oral administration, inhalation, transdermalapplication, or rectal administration. Depending on the route ofadministration, the active compound can be coated in a material toprotect the compound from the action of enzymes, acids and other naturalconditions which may inactivate the compound. For example, to administerB7-4 or PD-1 modulating agent by other than parenteral administration,it may be desirable to coat the peptide with, or co-administer thepeptide with, a material to prevent its inactivation.

A B7-4 or PD-1 modulating agent can be administered to an individual inan appropriate carrier, diluent or adjuvant, co-administered with enzymeinhibitors or in an appropriate carrier such as liposomes.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Adjuvant is used in its broadest sense and includes anyimmune stimulating compound such as interferon. Adjuvants contemplatedherein include resorcinols, non-ionic surfactants such aspolyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzymeinhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEEP) and trasylol. Liposomes includewater-in-oil-in-water emulsions as well as conventional liposomes(Sterna et al. (1984) J. Neuroimmunol. 7:27).

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringeability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating activecompound (e.g., a B7-4 or PD-1 polypeptide) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient (e.g., peptide) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

When the active compound is suitably protected, as described above, theprotein can be orally administered, for example, with an inert diluentor an assimilable edible carrier. As used herein “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the therapeutic compositions iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

In one embodiment of the present invention a therapeutically effectiveamount of an antibody to a B7-4 or PD-1 protein is administered to asubject. As defined herein, a therapeutically effective amount ofantibody (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kgbody weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an antibody can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with antibody in the range ofbetween about 0.1 to 20 mg/kg body weight, one time per week for betweenabout 1 to 10 weeks, preferably between 2 to 8 weeks, more preferablybetween about 3 to 7 weeks, and even more preferably for about 4, 5, or6 weeks. It will also be appreciated that the effective dosage ofantibody used for treatment may increase or decrease over the course ofa particular treatment. Changes in dosage may result from the results ofdiagnostic assays as described herein.

Monitoring the influence of agents (e.g., drugs or compounds) on theexpression or activity of a B7-4 or PD-1 protein can be applied not onlyin basic drug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to increase B7-4 or PD-1 gene expression, protein levels, orupregulate B7-4 or PD-1 activity, can be monitored in clinical trials ofsubjects exhibiting decreased B7-4 or PD-1 gene expression, proteinlevels, or downregulated B7-4 or PD-1 activity. Alternatively, theeffectiveness of an agent determined by a screening assay to decreaseB7-4 or PD-1 gene expression, protein levels, or downregulate B7-4 orPD-1 activity, can be monitored in clinical trials of subjectsexhibiting increased B7-4 or PD-1 gene expression, protein levels, orupregulated B7-4 or PD-1 activity. In such clinical trials, theexpression or activity of a B7-4 or PD-1 gene, and preferably, othergenes that have been implicated in a disorder can be used as a “readout” or markers of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including B7-4 orPD-1, that are modulated in cells by treatment with an agent (e.g.,compound, drug or small molecule) which modulates B7-4 or PD-1 activity(e.g., identified in a screening assay as described herein) can beidentified. Thus, to study the effect of agents on a B7-4 or PD-1associated disorder, for example, in a clinical trial, cells can beisolated and RNA prepared and analyzed for the levels of expression ofB7-4 or PD-1 and other genes implicated in the B7-4 or PD-1 associateddisorder, respectively. The levels of gene expression (i.e., a geneexpression pattern) can be quantified by Northern blot analysis orRT-PCR, as described herein, or alternatively by measuring the amount ofprotein produced, by one of the methods as described herein, or bymeasuring the levels of activity of B7-4 or PD-1 or other genes. In thisway, the gene expression pattern can serve as a marker, indicative ofthe physiological response of the cells to the agent. Accordingly, thisresponse state can be determined before, and at various points duringtreatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a B7-4 or PD-1protein, mRNA, or genomic DNA in the pre-administration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression or activity of the B7-4 or PD-1protein, mRNA, or genomic DNA in the post-administration samples; (v)comparing the level of expression or activity of the B7-4 or PD-1protein, mRNA, or genomic DNA in the pre-administration sample with theB7-4 or PD-1 protein, mRNA, or genomic DNA in the post administrationsample or samples; and (vi) altering the administration of the agent tothe subject accordingly. For example, increased administration of theagent may be desirable to increase the expression or activity of B7-4 orPD-1 to higher levels than detected, i.e., to increase the effectivenessof the agent. Alternatively, decreased administration of the agent maybe desirable to decrease expression or activity of B7-4 or PD-1 to lowerlevels than detected, i.e. to decrease the effectiveness of the agent.According to such an embodiment, B7-4 or PD-1 expression or activity canbe used as an indicator of the effectiveness of an agent, even in theabsence of an observable phenotypic response.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing, areincorporated herein by reference.

EXAMPLES Example 1 Isolation of B7-4 cDNA Molecules

The protein sequence of the extracellular domain of human B7-1 was usedto search the public databases for nucleic acid molecules encodinghomologous polypeptides. Two overlapping sequences in the EST database,AA292201 and AA399416, were identified. These sequences were used toisolate full-length B7-4 cDNAs from human activated keratinocyte andplacental cDNA libraries as follows.

Oligonucleotides with the sequence 5′-CAGCTATGGTGGTGCCGACTACAA-3′ (SEQID NO:5) and 5′-AGGTGCTAGGGGACAGTGTTAGACA-3′ (SEQ ID NO:6) from theseESTs were synthesized. These oligonucleotides were used to prime a PCRreaction using as template cDNA prepared by reverse transcription ofmRNAs from the spleen of a case of follicular lymphoma, activated Bcells, INF-γ activated keratinocytes, normal spleen, and placenta.Conditions were 94° C., 1 min; 94° C., 30 sec, 56° C., 30 sec, 68° C. 1min for 35 cycles; 68° C., 3 min, hold 4° C. All templates gave a bandof the expected size of 389 bp. The 389 bp product from the PCR of INF-γactivated keratinocytes was purified by agarose gel electrophoresis and0.12 ng was used as a template in a PCR reaction containing 0.05 mMbiotin-21-dUTP and the above primers. Conditions were 94° C., 1 min; 94°C., 30 sec, 56° C., 30 sec, 68° C., 2 min for 20 cycles; 68° C., 5 min,hold 4° C. The biotinylated PCR product was purified on a Nucleospincolumn (Clontech) and used as a probe in the ClonCapture cDNA selectionprocedure (Clontech). 60 ng of denatured, biotinylated PCR product wasincubated with 2 mM CoCl₂, 1×RecA buffer, 1 μg of RecA protein, 1×ATP ina final volume of 30 μl. The reaction was incubated at 37° C. for 15min. To that mixture, 0.7 μg of plasmid DNA of an activated keratinocytecDNA library and 0.4 μg of a human placental cDNA library was added andincubation continued for 20 min. 50 ng of EcoRV digested lambda DNA wasadded to the reaction and incubated 5 min. 0.6 μl of 10% SDS and 5.6 μgof proteinase K were added and incubated at 37° C. for 10 min.Proteinase K was inactivated by adding 1 μl of 0.1 M PMSF. Streptavidinmagnetic beads were preincubated with 5 μg of sheared salmon sperm DNAfor 10 min and the beads captured with a magnet, the supernatantremoved, and the beads resuspended in 30 μl of binding buffer (1 mMEDTA, 1 M NaCl, 10 mM Tris-HCl, pH 7.5). The beads were added to thereaction and the reaction incubated for 30 min at room temperature withgentle mixing. The beads were captured with a magnet and the supernatantremoved. The beads were washed with 1 ml of washing buffer (1 mM EDTA, 2M NaCl, 10 mM Tris-HCl, pH 7.5), beads were captured with a magnet andthe supernatant removed. The wash procedure was repeated 3 times. One mlof sterile H₂O was added to the washed beads, incubated 5 min at 37° C.,beads were captured on a magnet and the supernatant removed. CapturedDNA was eluted by adding 0.1 ml of elution buffer (1 mM EDTA, 0.1 NNaOH)., incubating 5 min at room temperature, beads were captured with amagnet and the supernatant removed and saved in a new tube. 22.5 μl ofprecipitation mix containing carrier and pH neutralizers was added alongwith 2.5 volumes of ethanol. The plasmid DNA was concentrated bycentrifugation and re-dissolved in H₂O. Plasmid DNA was re-introducedinto E. coli DH10B/P3 by electroporation and selected on LB-agar platescontaining 7.5 μg/ml tetracycline and 25 μg/ml ampicillin. Colonies werelifted onto Nytran filters and hybridized with ³²P-labeledoligonucleotides with the sequence 5′-CAGCTATGGTGGTGCCGACTACAA-3′ (SEQID NO:7), 5′-AGGTGCTAGGGGACAGTGTTAGACA-3′ (SEQ ID NO:8), and5′-TCGCTTGTAGTCGGCACCACCATA-3′ (SEQ ID NO:9). All oligos are fromAA292201 sequence. Final wash conditions were 2×SSC, 0.1% SDS at 55° for20 min. The two hybridizing colonies were picked and the sequence of thecDNA inserts was determined.

Sequencing revealed two forms of B7-4 molecules. The first form, B7-4secreted (B7-4S) encodes a protein having a short hydrophilic domainwithout a membrane anchor. The nucleotide and amino acid sequences ofthis form are shown in SEQ ID NO:1 and 2, respectively. The second form,B7-4 membrane (B7-4M) encodes a protein having a transmembrane and shortcytoplasmic domain. The nucleotide and amino acid sequences of this formare shown in SEQ ID NO:3 and 4, respectively. Both members of the B7-4family identified have signal, IgV, and IgC domains, as illustrated inFIGS. 3 and 4. The B7-4M form has approximately 21% amino acid identityto human B7-1 and about 20% amino acid identity to human B7-2 ascalculated using the default Blosum62 matrix with gap penalties set atexistence 11 and extension 1 (See the NCBI website), under conditionswhere B7-1 and B7-2 have about 26% identity.

Example 2 Expression of B7-4 mRNA: Northern Blot Analysis

An mRNA of the soluble form of B7-4 is predicted to be about 1.2 kbthough other sizes are possible. The mRNA of the second form is about3.8 kb, with minor mRNAs of 1.6 and 6.5 kb.

Expression of B7-4 polypeptides was analyzed. RNA was prepared byguanidine thiocyanate homogenization and cesium chloride centrifugation.Equal amounts of RNA (approximately 2 μg poly(A)+RNA) wereelectrophoresed on an agarose gel, blotted, and hybridized to a portionof ³²P-labeled B7-4 cDNA common to both the B7-4S and B7-4 M forms.These B7-4 mRNAs are highly expressed in placenta, lung, and heart andare moderately expressed in the thymus. In addition, these B7-4 mRNAsare weakly expressed in skeletal muscle, kidney, pancreas, prostate,testis, ovary, small intestine, colon, and peripheral blood leukocytes.They were also found to be very weakly expressed in liver or brain. B7-4mRNAs were not expressed in unstimulated monocytes, but were stronglyinduced by IFN-γ. Similarly, the expression of these polypeptides wasfound to be induced in keratinocytes by TPA/IFN-γ and in dendritic cellsby IFN-γ. These B7-4 mRNAs were not expressed in unstimulated B cells,but were induced by Ig crosslinking.

Expression of these B7-4 mRNAs was also examined in a variety of celllines. They were not found to be expressed in B cell lines such as Raji,Ramos, LBL, Nalm 6, and DHL-4. They were also not expressed in T celllines, such as Jurkat, Rex, CEM, HPB-ALL, Peer4, and H9 or in HTLV-1transformed T cell lines such as SPP and MT2 or in the myeloid lineU937.

Example 3 Further Characterization of B7-4 mRNA Expression: NorthernBlot Analysis

Mouse and human multiple tissue Northern blots (Clontech, Palo Alto,Calif.) were probed with ³²P-dCTP radiolabeled cDNA probes in QuikHyb(Stratagene, La Jolla, Calif.) according to the manufacturer'sinstructions. The human B7-4 probe consisted of a 1 kb BamHI/NotIfragment of the cDNA spanning the coding region and 3′ untranslatedregion of SEQ ID NO:1. The mouse B7-4 probe consisted of a 300 bp cDNAfragment from the coding region. Control actin probes were supplied byClontech. Blots were washed twice at room temperature in 2×SSC, 0.1%SDS, followed by 0.2×SSC, 0.1% SDS at 65° C., and examined byautoradiography.

B7-4 mRNA was expressed at high levels in heart, human placenta, andhuman fetal liver, and at lower levels in spleen, lymph nodes, thymus,and mouse liver.

B7-4 mRNA was expressed in a variety of transformed mouse cell lines,including PU5-1.8, RAW 264.7, K-Balb, M-MSV-Balb/3T3, Hepa 1-6, R1.1,L1210, P38D1, P815, and NB41A3 cells.

Example 4 Further Characterization of B7-4 mRNA Expression: QuantitativePCR, Genechip Hybridization, and RNA Blot Analysis

B7-4 mRNA expression on antigen presenting cells was examined andcompared to the expression of B7-1 and B7-2 on those cells. Forquantitative PCR analysis, cellular RNA was deoxyribonuclease-treated,re-extracted and converted to first strand cDNA. FAM(6-caroxyfluorescein)-labeled human B7-4, B7-1, B7-2, and GAPDH probeswere purchased from PE Biosystems (B7-4: primers5′-GCCGAAGTCATCTGGACAAG-3′ (SEQ ID NO:13) and 5′-TCTCAGTGTGCTGGTCACAT-3′(SEQ ID NO:14), probe 5′-FAM-CACCACCACCAATTCCAAGA-3′ (SEQ ID NO:15);B7-1: primers 5′-ACGTGACCAAGGAAGTGAAAGAA-3′ (SEQ ID NO:16) and5′-TGCCAGCTCTTCAACAGAAACAT-3′ (SEQ ID NO:17), probe5′-FAM-TGGCAACGCTGTCCTGTGGTCAC-3′ (SEQ ID NO:18); B7-2: primers5′-GGGCCGCACAAGTTTTGAT-3′ (SEQ ID NO:19) and5′-GCCCTTGTCCTTGATCTGAAGA-3′ (SEQ ID NO:20), probe5′-FAM-CGGACAGTTGGACCCTGAGACTTCACA-3′ (SEQ ID NO:21).

PCR reactions were set up in 96-well plates using reagents from thePerkin Elmer TaqMan™ EZ kit, according to the manufacturer'sinstructions. Standard curves were set up for each of the four genesanalyzed. Forty cycles of PCR were run in an ABI Prism 7700 SequenceDetector and GAPDH was used to normalize the B7-4, B7-1, and B7-2results.

The Affymetrix Mu19KsubA chip was used for Genechip hybridizationanalysis. The sequence of a portion of murine B7-4 is represented byexpressed sequence tag TC 17781 of The Institute for Genomic Research onthis chip. RNA isolation, chip hybridization and scanning was performedas described in Byrne, M. C. et al. (2000) Curr. Prot. Mol. Biol. Suppl.49:22.2.1-22.2.13.

For RNA blot hybridization, the 1.6 kb human and 3.6 kb murine B7-4cDNAs were excised by digestion with Xba I and labeled by random primingwith γ-³²P-ATP and the Klenow fragment of DNA polymerase I. RNA blotswere hybridized as described in Freeman, G. J. et al. (1992) J. Immunol.149:3795-3801.

Human dendritic cells were derived from peripheral blood. Mononuclearcells were isolated after fractionation on a Ficoll gradient.Non-adherent cells were removed and the remaining cells cultured in 150ng/ml human GM-CSF (R&D Systems) and 100 ng/ml human IL-4 (R&D Systems)for 2 days. The non-adherent dendritic cells were isolated (CD80⁺ CD86⁺HLA-DR⁺ CD54⁺ CD58⁺ CD1a⁺) and cultured in GM-CSF alone or activatedwith GM-CSF, 2.5 μg/ml LPS (Sigma Chemicals), and 10 ng/ml humanInterferon-γ. At 4 hours and 20 hours after activation, cells wereharvested and RNA isolated using the RNeasy kit (Qiagen).

Murine bone marrow mononuclear cells were immuno-depleted ofgranulocytes, lymphocytes and Ia⁺ cells by magnetic activated cellsorting and cultured in petri dishes with GM-CSF and IL-4. Dendriticcells were harvested as the non-adherent population after 7 days ofculture, and demonstrated to be 75-80% CD11c⁺, high IA⁺ cells. Cellswere activated with LPS and human interferon-γ.

Analysis of expression in human blood monocytes by RNA blothybridization demonstrated that B7-2 is not expressed by unstimulatedmonocytes, but is rapidly upregulated upon interferon-γ treatment.Treatment of monocytes with another pro-inflammatory cytokine, tumornecrosis factor (TNF)-α led to a low level induction similar to thatfound with medium alone, presumably as a result of activation byadherence to plastic. In addition to the major 4.2 kb B7-4 mRNA, a minor1.8 kb B7-4 mRNA species was also observed in interferon-γ treatedmonocytes. Expression of B7-4 by human B-cells activated by cell surfaceimmunoglobulin cross-linking, but not by the Raji cell line, was alsoobserved. Similarly, B7-1 is not expressed by unstimulated monocytes,but is upregulated in response to interferon-γ with kinetics similar toB7-4 expression. In contrast, B7-2 mRNA is constitutively expressed inmonocytes and levels are unaffected by interferon-γ or TNF-α treatment.

B7-4, B7-1, and B7-2 mRNA expression by human dendritic cells was alsoexamined by quantitative PCR. Human peripheral blood-derived dendriticcells were treated with granulocyte-macrophage colony stimulated factor(GM-CSF) alone or activated with GM-CSF, lipopolysaccharide (LPS), andinterferon-γ. As a result of activation by LPS and interferon-γ, B7-4mRNA was rapidly induced with a 16-fold increase at 4 hours and a34-fold increase at 20 hours, relative to non-induced cells. B7-1 andB7-2 mRNAs were also induced upon activation: B7-1 was induced 21-foldat 4 hours and 22-fold at 20 hours. B7-2 showed little induction at 4hours; however, expression was induced 5-fold at 20 hours. Expression ofB7-4 by murine bone marrow-derived dendritic cells treated with LPS andinterferon-γ was examined using Genechip™ hybridization. B7-4 expressionin these cells follows a pattern similar to that observed on humandendritic cells: a 5-fold induction of the B7-4 mRNA relative to theuninduced cells at 6 and 20 hours after induction. These datademonstrate that B7-4 is expressed by antigen presenting cells andlymphocytes, and it is induced on dendritic cells in a manner similar toB7-1 and B7-2. Treatment of human keratinocytes with phorbol ester andinterferon-γ also induced B7-4.

In murine tissues, an approximately 3.7 kb B7-4 mRNA transcript wasdetected by northern blot hybridization. The distribution of the murineB7-4 mRNA closely resembled that of the human B7-4, with high levels inheart, thymus and lung, and low levels in kidney, spleen and liver.

Example 5 Chromosomal Localization of B7-4

The chromosomal localization of the B7-4 genes was determined using amonochromosomal blot kit commercially available from Quantum (Toronto,Canada). The blots were probed with a sequence that recognizes bothB7-4S and B7-4M. Using this method, the B7-4 polypeptides have beenlocalized to human chromosome 9, whereas B7-1 and B7-2 have beenlocalized to human chromosome 3. The butyrophilins, which also sharelimited amino acid sequence identity with the B7-4 family have beenlocalized to the major histocompatability complex on chromosome 6. Thechromosomal location of B7-4 was confirmed using B7-4 specific primersin PCR amplification of monochromosomal somatic cell hybrid DNAtemplates available from Quantum Technologies (Canada).

Example 6 Binding of B7-4 Molecules to T Cell Ligands or Antibodies

COS cells were transfected with either vector DNA (pcDNAI), or anexpression plasmid containing the B7-4M cDNA. After 72 hours, thetransfected COS cells were detached by incubation in PBS containing 0.5mM EDTA for 30 min. at 37° C.

The ability of COS cells expressing B7-4M to bind to various T cellreceptors and antibodies was tested. FACS analysis of binding of CD28Ig,CTLA4-Ig, and control Ig by B7-4-transfected COS cells showed thatneither CD28Ig nor CTLA4-Ig was bound by B7-4 (FIG. 8). The ability ofCOS cells expressing B7-4M to bind to IgG and murine ICOS-his fusionprotein was also tested. No binding of human B7-4 to murine ICOS wasdetected (FIG. 9). As shown in FIG. 10, FACS analysis revealed bindingof BB1 (anti B7-1 and anti B7-3), but not IgM or 133 (anti-B7)antibodies to B7-4-transfected COS cells.

Example 7 Costimulation of T Cell Proliferation By B7-4 Molecules

The ability of B7-4 polypeptides to costimulate human T cellproliferation was tested.

Human CD28⁺ T cells were isolated by immunomagnetic bead depletion usingmonoclonal antibodies directed against B cells, natural killer cells andmacrophages as previously described (Gimmi, C. D., et al. (1993) Proc.Natl. Acad. Sci. USA 90, 6586-6590). B7-4 and vector transfected COScells were harvested 72 hours after transfection, incubated with 25μg/ml of mitomycin-C for 1 hour, and then extensively washed. 10⁵ naïveT Cells were stimulated with plate bound anti-CD3 mAb plus 20,000mitomycin-c treated COS cells transfected with the indicated DNAconstruct.

T cell proliferation was measured by 3H-thymidine (1 μCi) incorporatedfor the last 12 hours of a 72 hour incubation. As shown in FIGS. 11 and12, COS cells expressing B7-4 can costimulate T cell proliferation.

Example 8 Generation of Murine Antibodies to B7-4

Mammalian expression vectors (pEF6 or pcDNA3.1 (Invitrogen)) wereprepared comprising the entire murine or human B7-4 cDNA. ThecDNA/vector construct was dissolved in 0.9% saline at 1 mg/ml (not TE orPBS).

Before immunization, 78 μl of 1 mg/ml cardiotoxin (Sigma #C-1777) in0.9% saline was injected into the tibialis anterior muscle of each hindlimb of the mouse being immunized. Each mouse was then left alone for 5days.

After anesthetizing the mice, 50 μl of 1 mg/ml purified B7-4 cDNA/vectorconstruct (in 0.9% saline) was injected into each regenerating tibialisanterior muscle.

Antibody titers were measured approximately six days after immunizationusing standard methods, for example, in an ELISA assay. The cDNAimmunization was repeated every 2-4 weeks for three cycles (until theantibody titre was>1:10,000). Mice were then boosted with CHO cellstransfected with PDL-1.

Spleen cells isolated from mice having appropriate antibody titers wereharvested. The spleen cells were fused to fusion partners SP2-0) to makehybridomas. Hybridomas and antibodies were manipulated using standardmethods (see, e.g., “Antibodies: A Laboratory Manual”, Harlow, E. andLane, D., Cold Spring Harbor Laboratory (1988), which is incorporatedherein by reference).

Antibodies 2A3, 10D9, 5A9, and 11D12 were among those selected inscreening assays. These antibodies were found to bind to COS or CHOcells transfected with human B7-4 and not to mock transfected cells orto cells transfected with mouse B7-4. The antibodies were used to detectthe presence of B7-4 on various cell populations. B7-4 expression wasobserved, inter alia, on heart tissue, tumor cells (including some lungtumor cells, some ovarian tumor cells, some breast tumor cells, someepithelial tumor cells, and some squamous cell carcinomas), placenta,and thymic epithelium.

Example 9 Generation of Fully Human Antibodies to B7-4

In this example, fully human antibodies against B7-4 or PD-1 are made inmice that are transgenic for human immunoglobulin genes. Transgenic miceare made using standard methods, e.g., according to Hogan, et al.,“Manipulating the Mouse Embryo: A Laboratory Manual”, Cold Spring HarborLaboratory, which is incorporated herein by reference, or are purchasedcommercially. Embryonic stem cells are manipulated according topublished procedures (Teratocarcinomas and embryonic stem cells: apractical approach, Robertson, E. J. ed., IRL Press, Washington, D.C.,1987; Zjilstra et al. (1989) Nature 342:435-438; and Schwartzberg et al.(1989) Science 246:799-803, each of which is incorporated herein byreference). DNA cloning procedures are carried out according toSambrook, J. et al. in Molecular Cloning: A Laboratory Manual, 2d ed.,1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,which is incorporated herein by reference. Oligonucleotides aresynthesized, e.g., on an Applied Bio Systems oligonucleotide synthesizeraccording to specifications provided by the manufacturer or arepurchased commercially.

Transgenic mice are immunized using a purified or recombinant B7-4 orPD-1 or a fusion protein comprising at least an immunogenic portion ofthe extracellular domain of B7-4 or PD-1. Approximately four hundred μgof B7-4 or PD-1 in 100 μL of phosphate buffered saline (PBS) is injectedintraperitoneally into each mouse. Serum samples are collectedapproximately six days later by retro-orbital sinus bleeding.

Antibody reactivity and specificity for B7-4 or PD-1 are assessed usingan indirect enzyme-linked immunosorbent assay (ELISA). Severalimmunoglobulin superfamily molecules are tested as controls (e.g., CTLA4and CD28) to analyze the antibody specificity of the antibody for B7-4or PD-1. Antibodies having human variable regions which bind to B7-4 orPD-1 are detected by enzyme conjugates specific for human IgM and humanIgG sub-classes with no cross reactivity to mouse immunoglobulin.Briefly, PVC microtiter plates are coated with B7-4 or PD-1 by coatingwells overnight at 37° C. with 5 μg/mL B7-4 in PBS. Serum samples arediluted in PBS, 5% serum, 0.5% Tween-20 and are incubated in the wellsfor 1 hour at room temperature, followed by anti-human IgG Fc and IgGF(ab′)-horseradish peroxidase or anti-human IgM Fc-horseradishperoxidase in the same diluent. After 1 hour at room temperature enzymeactivity is assessed by addition of ABTS substrate (Sigma, St. Louis,Mo.) and read after 30 minutes at 415-490 nm. In pre-immunization serumsamples from the same mice, titers of human antibodies to the sametarget antigens are also tested.

Spleen cells isolated from mice having appropriate antibody titers areharvested. The spleen cells are fused to appropriate fusion partners(e.g., myeloma cells) to make hybridomas. Hybridomas and antibodies aremanipulated according to “Antibodies: A Laboratory Manual”, Ed Harlowand David Lane, Cold Spring Harbor Laboratory (1988), which isincorporated herein by reference.

The complementarity determining sequences of the murine VH and VLdomains of a murine antibody could be used to graft into the frameworkof human immunoglobulins in order to generate a humanized antibodyagainst B7-4 or PD-1 (Riechmann et al. 1988. Nature 332:323; Verhoeyenet al. 1988. Science 239:1534).

Example 10 Generation of Human Single Chain Fvs Reactive with B7-4 orPD-1

As an alternative to preparing monoclonal antibody-secreting hybridomas,anti B7-4 or anti-PD-1 antibodies (single chain Fv-like portions ofantibodies) were identified and isolated by screening a combinatoriallibrary of human immunoglobulin sequences displayed on M13 bacteriophagefrom Cambridge Antibody Technology. Ltd., Melbourn, UK (Winter et al.1994 Annu. Rev. Immunol. 1994 12:433; Hoogenboom et al., 1998,Immunotechnology 4:1) PD-1.Fc or B7-4.Fc was used to thereby isolateimmunoglobulin library members that bind a B7-4 or PD-1 polypeptide.Kits for generating and screening phage display libraries arecommercially available and standard methods were employed to generatethe scFv (Helfrich et al. J. Immunol Methods 2000. 237: 131-45; Cardosoet al. Scand J. Immunol 2000. 51: 337-44.)PD-1.Fc or B7-4.Fc wereimmobilized on plastic and phage expressing specific scFv were selectedby panning and multiple rounds of enrichment (Griffiths et al. 1993 EMBOJ. 12:725).

Example 11 Identification of a receptor for B7-4

Fusion proteins consisting of the extracellular region of human PD-1fused to the hinge-CH2—CH3 domains of either human immunoglobulin gamma1 or murine Ig gamma2a (with mutations blocking FcR and complementinteraction) were used to search for a ligand that binds to PD-1. Aspart of this search, staining of the cell surface of monocytesstimulated with gamma-interferon was found. B7-4 is induced in monocytesafter stimulation with gamma-interferon, as observed by northern blothybridization.

The binding of PD-1-Fc (human Ig gammal) to the surface of COS cellstransiently transfected with a B7-4-expression vector was tested. COScells were transfected with either B7-4M or B7-1 using LipofectAMINEtransfection reagent. After 48 hours, the cells were stained with humanPD-1-Fc, murine PD-1-Fc, CTLA4-Fc, Flt4-Fc, or IgG followed by anti-IgGconjugated to phycoerythrin (PE). The cells were then analyzed by flowcytometry. As shown in FIG. 13, COS cells expressing B7-4 bound bothhuman PD-1-Fc and murine PD-1-Fc, but did not bind CTLA4-Fc, Flt4-Fc, orhuman IgG. As a positive control, it was demonstrated that B7-1expressing COS cells bound CTLA4-Fc, but not PD-1-Fc or IgG.

In addition, an in situ assay of transfected COS cell monolayers wasperformed. Monolayers were probed with PD-1Fc, CTLA4Fc or human IgG1 andbinding was detected with a secondary antibody directed against the Fcportion and conjugated to alkaline phosphatase. Binding was visualizedwith chromogenic substrates 5-bromo-4-chloro-3-indolyl phosphate andnitro blue tetrazolium and light microscopy. In parallel, cellstransfected with B7-4 were found to bind to PD-1-Fc, and not CTLA4-Fc(human Ig gamma 1) or Flt4-Fc, the extracellular region of murine Flt4linked to human Ig gamma 1. In parallel, PD-1Fc did not bind the surfaceof mock-transfected, B7-1 or B7-2 transfected COS cells.

In another experiment, no binding of PD-1-Fc to soluble forms of B7-1 orB7-2 and binding to B7-4 was detected using a BIACORE-based assay. Inparallel, hCTLA4 was shown to bind to B7-1 and not to B7-4. PD-1-Fc orCTLA4-Fc was immobilized and conditions were essentially as described byFitz et al. (1997) Oncogene 15:613). Concentrated COS cell medium fromcells that had been transfected with full length B7-4M or B7-4-Fc wasinjected and interactions were measured using real-time BiomolecularInteraction Analysis (BIA) (Sjolander, S. and Urbaniczky, C. (l 991)Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol. 5:699-705). Human B7-4 was found to bind human and mouse PD-1 andthis binding was inhibited by competition with a coinjected PD-1-Fc, butnot CTLA4-Fc. These experiments demonstrate not only the binding ofsoluble B7-4-Fc fusion protein to immobilized PD-1-Fc, but alsodemonstrate the presence of a soluble form of B7-4 in the conditionedmedium of B7-4M cDNA transfected cells, presumably as a result ofshedding.

FIG. 14 illustrates the ability of PD-1 and not Flt4 (the receptor forvascular endothelial growth factor C) to competitively inhibit thebinding of PD-1 to B7-4. The binding of human PD-1 gamma 2a fusionprotein to COS cells expressing B7-4M is shown in Panel A. The bindingwas detected with anti-gamma 2a specific reagents linked to PE. HumanPD-1 linked to IgGI was added at: 50 μg/ml, 6.25 μg/ml, 100 μg/ml, or 25μg/ml and was found to compete for binding. As a control, Flt4IgG1 at100 μg/ml was not found to compete for binding of PD-1 to B7-4.

In yet another experiment, the ability of B7-4 to bind to PD-1 wasdetermined by flow cytometry and BIACORE-binding assays. Human andmurine PD-1.Ig fusion proteins bound to both human and murine B7-4expressed on CHO cells, as detected by flow cytometry (FIG. 15).However, neither human CTLA-4.Ig, human CD28.Ig, nor human ICOS.Ig boundto either B7-4 expressing cell line. The PD-1 fusion proteins did notbind CHO cells transfected with vector alone. Further confirmation ofthe PD-1:B7-4 interaction was obtained using surface plasmon resonancewith a BIACORE instrument. The human and murine PD-1.Ig proteins andhuman CTLA-4.Ig were immobilized on the flow cell surfaces of a dextranchip and tested for binding to soluble human B7-4.Ig. B7-4.Ig bound toboth human and murine PD-1.Ig, but not to human CTLA-4.Ig (FIG. 16).This binding was blocked by competition with co-injected solublePD-1.Ig, but not CTLA-4.Ig. Soluble forms of human B7-1 and B7-2 did notbind immobilized human PD-1.

These data demonstrate that PD-1 binds B7-4, and that this interactionmay regulate the action of PD-1.

Example 12 B7-4 Can Transmit a Negative Signal to Immune Cells

In this example, 5×10⁵ Jurkat T cells per well were stimulated withanti-CD3 coated beads (at a 1:1 ratio) and soluble anti-CD28. COS cellsexpressing B7-4 or a negative control, called EZZ, were titrated intothe wells. Supernatants were harvested at 48 hours and assayed by ELISAfor human IL-2. FIG. 17 shows that increasing numbers of COS B7-4 cells(bars on the right in the figure) lead to a decrease in IL-2 production.

Using similar assay formats, for example in which human PHA-blasts fromPBMCs were stimulated with immobilized anti-CD3 and soluble anti-CD28, adecrease in T-cell proliferation was observed by titrating in COS cellsexpressing B7-4.

Example 13 The PD-1:B7-4 Interaction Inhibits CD3-mediated T-CellProliferation

To examine the functional significance of the PD-1:B7-4 interaction, thefunctional consequences of B7-4 interaction with its receptor were alsoexamined using human T-cells. Peripheral blood mononuclear cells wereisolated by Ficoll-Hypaque gradient centrifugation. CD4⁺ T cellpopulations (85-90% purity) were purified by negative selection using acocktail of monoclonal antibodies and immunomagnetic beads(PerseptiveBiosystems). Anti-CD3, control IgG and fusion protein werecovalently attached to polyurethane-coated tosyl activated Dynabeads(Dynal) according to manufacturer's instructions and as describedpreviously (Blair, P. J. et al. (1998) J. Immunol. 160:12-15). Anti-CD3antibody (UCHT1, Pharmingen) at the indicated concentration was added to1×10⁷ beads/ml 0.1 M phosphate buffer pH 7.4. Control IgG was added tothe bead suspension in order to maintain a constant total Igconcentration of 5 μg/ml during binding. Similarly,anti-CD3/B7-4.Ig(γ2a) beads were prepared with the indicated anti-CD3antibody concentration, a constant concentration of either B7-4.Igrepresenting 40% of the total bound protein (2 μg/10⁷ beads), andcontrol IgG to make up the remaining total bound protein. 10⁵T cellswere cultured in 96 well flat-bottom plates, and beads were added at a 1bead to 1 cell ratio in the presence or absence of the indicatedconcentrations of anti-CD28 antibody (CD28.2, Pharmingen). Proliferationwas determined by labeling cultures for the last 6 hr of a 4-day assaywith 1 μCi ³H-thymidine/well. For analysis by cytokine ELISAs, cultureswere set up as described above and supernatants harvested at theindicated times. Interferon-γ, IL-10 and IL-2 concentrations weredetermined using commercially available ELISA kits (Genzyme).

Purified CD4⁺ T-cells obtained from peripheral blood mononuclear cells(PBMC) were activated with beads coated with anti-CD3 mAb and eitherhuman B7-4.Ig or a control Ig. Proliferation and cytokine production wasassessed 96 hours after stimulation. As shown in FIG. 18, cellsactivated with anti-CD3 mAb/B7-4.Ig coated beads showed a 69% decreasein proliferation relative to anti-CD3 mAb/control Ig activated cells.Furthermore, activation of cells in the presence of B7-4 also impairedcytokine secretion. In the presence of B7-4, interferon-γ and IL-10secretions were decreased by approximately 80% and 60%, respectively(FIG. 18). IL-2 production was below detection under these activationconditions at both 24 and 96 hr. However, under conditions in whichcostimulation in the form of soluble anti-CD28 was provided, activationof cells in the presence of B7-4 also led to a decrease in IL-2production. Thus, activation of murine and human T-cells in the presenceof B7-4 leads to inhibition of both proliferation and cytokinesecretion.

Example 14 The Outcome of PD-1:B7-4 Interaction Depends on the Strengthof T-cell Receptor and CD28 Signals

To examine the relationship between T-cell receptor, CD28 and PD-1mediated signals, human CD4⁺ T-cells were stimulated with suboptimal oroptimal concentrations of anti-CD3 mAb, a fixed concentration of B7-4.Igand increasing concentrations of soluble anti-CD28 mAb. Using anti-CD3mAb-coated beads, the concentrations required for suboptimal and optimalT-cell stimulation were established. Under conditions of suboptimalT-cell receptor engagement (anti-CD3 mAb at 1 μg/ml), minimalproliferation was observed in the absence of costimulation (FIG. 19A).Addition of increasing concentrations of soluble anti-CD28 mAb led to anup to 30-fold increase in proliferation. Under these conditions,activation of T cells in the presence of B7-4 resulted in an 80%reduction in proliferation (FIG. 19A). A maximal level of costimulation(anti-Cd28 at 250 ng/ml) was required to rescue the inhibition ofproliferation mediated by B7-4 stimulation. In contrast, undersaturating conditions of T-cell receptor activation (anti-CD3 mAb at 2μg/ml), B7-4 mediated inhibition of T-cell proliferation was onlyobserved in the absence of CD28 costimulation (FIG. 19B).

Example 15 Ability of B7-4 To Inhibit CD28 Signals and CytokineProduction

The inhibitory effects of the PD-1:B7-4 pathway appear to be determinedby the strength of signal through the TCR and CD28 (see previousexample), whereby weak CD3/CD28-mediated responses are easilydownregulated. To study the interaction of the CD28 signal and thePD-1:B7-4 pathway, pre-activated DO11.10 CD4⁺ T cells were activatedwith OVA peptide presented by CHO-IA^(d)/B7.2 or CHO-IA^(d)/B7.2/B7-4.

For detection of B7-4, 5×10⁴ CHO transfectants cells were incubated with5 μg/ml of human PD-1Ig (hPD-1-Ig) (Genetics Institute, Cambridge,Mass.) and developed with goat anti-murine IgG2a-phycoerythrin (PE)(Southern Biotechnology Associates Inc., Birmingham, Ala.). In addition,cells were stained separately with 5 μg/ml anti-IA^(d)-PE or B7.2-PE(Pharmingen, San Diego, Calif.). Following each step, cells were washedthree times with PBS/1% BSA/0.02% sodium azide. After the finalincubation, cells were fixed with 1% paraformaldehyde. Ten thousandevents were analyzed on a FACSCalibar (Becton Dickinson, Mountain View,Calif.). All isotype controls were all obtained from Pharmingen.

Splenocytes were prepared from DO11.10 mice and treated with Tris-NH₄Clto deplete erythrocytes. Cells were cultured with 1 μg/ml of OVA peptidefor 72 hours (Analytical Biotechnology Services, Boston, Mass.) in RPMI1640 (Life Technologies, Grand Island, N.Y.) supplemented with 10% FCS(Sigma, St Louis, Mo.), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/mlstreptomycin, 250 ng/ml amphotericin B, 10 mM HEPES, 50 μM 2-ME (allfrom Life Technologies) and 15 mg/ml of gentamicin (BioWhittaker,Walkersville, Md.). CD4⁺ T cells were purified by positive selectionusing magnetic-activated cell sorting separation columns (MiltenyiBiotec, Auburn, Calif.) with resulting purity of >98%. Cells were restedovernight before re-stimulation.

Proliferation of CHO cells was inhibited by incubation with 50 μg/ml ofmitomycin C (Bristol Laboratories, Princeton, N.J.) for 16 hours at 37°C. At the end of the incubation period, the cells were harvested with 10mM EDTA in PBS, washed twice and left on ice for 1 hour. The cells weresubsequently washed three times and resuspended in culture medium. 10⁵pre-activated CD4⁺ T cells were cultured with varying concentrations ofOVA peptide and 10⁴ mitomycin C-treated CHO transfectants in 96 wellplates. To assay proliferation, cultures were incubated for 48 hrs andpulsed with 1 μCi/well of [³H] thymidine (New England Nuclear, Boston,Mass.) for the last 6 hours of the incubation period.

The expression of B7 and IA^(d) was similar on all CHO transfectants(FIG. 20). As expected, introduction of B7.2 led to an increase inproliferative responses by T cells at all antigen concentrations (FIG.21). However, B7-4 inhibited responses at lower peptide concentrations(0.01 μg/ml and 0.001 μg/ml) (FIG. 21).

To address the capacity of PD-1:B7-4 pathway to inhibit cytokineproduction, supernatants from DO11.10 CD4⁺ T cells activated with OVApeptide presented by CHO cell transfectants were analyzed. Aliquots ofsupernatants were harvested at various times after initiation ofcultures. IL-2, IL-4, IFN-γ and IL-10 levels were analyzed using mAbsand recombinant cytokine standards from Pharmingen. Detection limitswere as follows: IL-2, 20 pg/ml, IL-4, 40 pg/ml. IFN-γ, 100 pg/ml andIL-10, 200 pg/ml. Production of IL-2, IL-4, IFN-γ and IL-10 wasinhibited significantly when DO11.10 CD4⁺ T cells were cultured with 0.1μg/ml peptide and B7-4 (FIG. 22). At this concentration there was only aweak inhibition of proliferation. However B7-4 significantly inhibitedcytokine production at 0.01μg/ml peptide, consistent with the inhibitionof proliferation (FIG. 23). IL-10 was not detected under theseactivation conditions. Therefore, PD-1 engagement by B7-4 candownregulate cytokine production even when T cell proliferation is notaffected.

To determine whether the diminished cytokine production was due toreduced mRNA levels, and RNase protection assay was utilized. CD4⁺ Tcells were restimulated with various CHO cell transfectants and 0.01μg/ml OVA peptide. After 48 hours, cells were harvested and mRNA wasisolated using TRIzol® reagent (Life Technologies). 5 μg mRNA wasanalyzed for cytokine levels by RNase protection assay using RiboQuantmultiprobe kit mCK1 according to the manufacturer's instructions(Pharmingen). Transcript levels of IL-4, IL-10, IL-13, IL-2, IL6 andIFN-γ mRNA were detected in pre-activated DO11-10 CD4⁺T cells afterstimulation with 0.01 μg/ml OVA peptide presented by CHO-IA^(d)/B7.2.However, the introduction of B7-4 significantly reduced cytokine mRNAlevels. There was minimal upregulation of mRNA for cytokines inunstimulated T cell cultures or T cells activated with peptide presentedby CHO-IA^(d). These results further demonstrate the capacity of thePD-1:B7-4 pathway to antagonize a strong B7/CD28 signal at least whenantigenic stimulation is weak or limiting, and the inhibition of atleast cytokine production in conditions of strong antigenic stimulation.

Example 16 Mechanism of Action of the PD-1:B7-4 Pathway

Cross-linking of CTLA-4 has been shown to inhibit cell cycle progressionin naïve T cells (Krummel, M. F. and Allison, J. P. (1996) J. Exp. Med.183:2533-2540; Walunas, T. L. et al. (1996) J. Exp. Med. 183:2541-2550).As PD-1 was isolated from murine cell lines undergoing apoptosis, apossible mechanism of action of the PD-1:B7-4 pathway might be toincrease programmed cell death. To address this issue, DO11.10 CD4⁺ Tcells were restimulated with 0.01 μg/ml peptide and various CHOtransfectants and cell cycle progression was analyzed. CD4⁺ T cells wererestimulated with 0.01 μg/ml peptide as described previously. After 36hours of culture, cells were recovered and stained with anti CD4-FITC.Cells were washed in PBS, fixed in 70% ethanol for 1 hour on ice andthen resuspended in PBS containing 10 μg/ml RNase (Sigma) and 50 μg/mlpropidium iodide (Sigma). Analysis was performed within an hour ofstaining.

After 48 hours, cells were recovered and stained with CD4-FITC. Afterpermeabilization, cells were incubated with propidium iodide to analyzethe G₀/G₁, S/G₂ and sub-diploid populations. CD4⁺ T cells restimulatedwith peptide presented by CHO-IA^(d) have a large proportion of cells inthe sub-diploid population, indicative of apoptosis (FIG. 24). Incultures where CD4⁺ T cells were stimulated by peptide presented byCHO-IA^(d)/B7-2, there were increased number of cells in the SG₂ phase,and a decreased number in the sub-diploid population, indicating thatcells were in cycle and rescued from apoptosis by B7/CD28 costimulation.The introduction of B7-4 led to an increased number of cells in theG0/G1 phase (FIG. 24). There were comparable levels of apoptosis in theB7-4 cultures to the CHO-IA^(d)/B7 cultures. This was confirmed byannexin staining. The inhibition of cell progression by the PD-1:B7-4pathway confirms its role in downregulating T cell activation.

Example 17 Inhibition of binding of biotinylated human B7-4 Fc to humanPD-1 Fc

Fc fusion proteins were generated by linking the extracellular region ofPD-1 or B7-4 to the hinge-CH2—CH3 domains of murine Igγ2a. Recombinantproteins were produced in COS cells transiently transfected withLipofectAMINE (Gibco-BRL) or stably transfected CHO cell lines andpurified from conditioned media using protein A-Sepharose.

The ability of antibodies to B7-4 or PD-1 to inhibit the interaction ofhuman B7-4Fc and human PD-1 Fc was tested using standard ELISA methods.Briefly, human PD-1 Fc molecules were immobilized in 96-well plates,blocked, and washed. Biotinylated B7-4Fc molecules (100 ng/ml) wereadded to wells at concentrations of approximately 2000, 700, 200, 70,25, 8, and 1.18 ng/ml (FIG. 25). The wells were incubated withStrepAvidin conjugated horse radish peroxidase, washed, and color wasdeveloped using standard methods. The ED50 of B7-4Fc was found to be 108ng/ml.

The ability of murine antibodies to human B7-4 (10D9 and 11D12) or scFvportions of human immunoglobulins (B7-4-1, B7-4-6, and B7-4-12) toinhibit the binding of biotinylated human B7-4Fc to human PD-1 Fc wastested at 7 concentrations of inhibitors. The IC50 was found to rangefrom 0.5 nM to 24 nM and the data are presented in FIG. 25.

The PD-1 specific scFv were also tested for their ability to inhibit thebinding of B7-4 Fc to PD-1 Fc using the same ELISA methods describedabove. Human scFv reactive with PD-1 (PD1-17 scFv) were found to inhibitspecific binding (EC50 between 10⁻⁷ and 10⁻⁸) as shown in FIG. 26. V_(L)and V_(H) domains of the PD1-17scFv were used to generate a completeIgG. In brief, the VH and VL coding regions were linked to genomic CHand CL gene sequences in expression vectors. The resulting expressionvectors were transiently transfected into human 293 cells and the IgGharvested from the conditioned medium. The potency of the grafted wholeIgG molecule was higher than for the scFv antibody (EC 50 between 10⁻⁸Mand 10⁻⁹M).

Example 18 Administration of Soluble B7-4Fc Exacerbates Disease in aMurine Model

To determine if modulation of the B7-4/PD-1 pathway has immunoregulatoryactivity in vivo, the protein was evaluated in a murine model ofexperimental autoimmune encephalomyelitis (EAE) that shares manyclinical and pathological features with the human disease multiplesclerosis. Female SJL/J mice were immunized with 100 μg of proteolipidprotein (PLP) in complete Freund's adjuvant. Ten days later, spleenswere harvested, processed to single cell suspensions and thenrestimulated in vitro with 5 μg of PLP for 96 hours. Cells were washedthree times in PBS and then 15×10⁶ cells transferred to naive SJL/J miceby intraperitoneal injection. The adoptive transfer of autoreactive Tcells results in acute paralysis of recipient mice which manifests asloss of tail tone with subsequent progression to full hind limbparalysis. This paralytic episode coincides with marked infiltration ofactivated T cells and macrophages in the CNS. Under most conditions,this is an acute model of disease with spontaneous recovery occurringafter a short period of paralysis. For evaluation of B7-4Fc, mice wereinjected subcutaneously with 200 μg of the protein in 100 μl of sterilesaline on days 0, 2, 4, 7 and 11 after cell transfer (n=10). Controlmice (n=10) received an equal volume of saline only. All animals weremonitored regularly for clinical signs of disease which were scored asfollows: 1. Loss of tail tone; 2. Hind limb weakness/partial hind limbparalysis; 3. Complete hind limb paralysis; 4. Hind and forelimbparalysis; 5. Moribund.

In the experiment shown in FIG. 27, the incidence and onset of clinicaldisease were similar in both groups. Mice treated with the B7-4Fchowever, developed severe disease with the majority of animals rapidlyprogressing to complete hind and forelimb paralysis (9/10 and 1/10 forB7-4Fc and control mice respectively). Mortality associated withclinical signs of disease was 10% in the control group and 70% in theB7-4Fc treated mice. In addition, recovery from clinical disease wassubstantially delayed in the B7-4Fc treated mice that did survivedespite the fact that treatment was discontinued on day 11.

In conclusion, using an adoptive transfer model of T cell mediatedautoimmunity, administration of soluble B7-4Fc exacerbates clinicalsigns of disease resulting in increased mortality and delayed recoveryfrom paralysis. These findings are consistent with enhancedactivation/infiltration of inflammatory cells into the CNS and clearlydemonstrate the immunoregulatory potential for the B7-4Fc protein invivo.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

23 1 968 DNA Homo sapiens CDS (59)..(793) 1 gcttcccgag gctccgcaccagccgcgctt ctgtccgcct gcagggcatt ccagaaag 58 atg agg ata ttt gct gtc tttata ttc atg acc tac tgg cat ttg ctg 106 Met Arg Ile Phe Ala Val Phe IlePhe Met Thr Tyr Trp His Leu Leu 1 5 10 15 aac gca ttt act gtc acg gttccc aag gac cta tat gtg gta gag tat 154 Asn Ala Phe Thr Val Thr Val ProLys Asp Leu Tyr Val Val Glu Tyr 20 25 30 ggt agc aat atg aca att gaa tgcaaa ttc cca gta gaa aaa caa tta 202 Gly Ser Asn Met Thr Ile Glu Cys LysPhe Pro Val Glu Lys Gln Leu 35 40 45 gac ctg gct gca cta att gtc tat tgggaa atg gag gat aag aac att 250 Asp Leu Ala Ala Leu Ile Val Tyr Trp GluMet Glu Asp Lys Asn Ile 50 55 60 att caa ttt gtg cat gga gag gaa gac ctgaag gtt cag cat agt agc 298 Ile Gln Phe Val His Gly Glu Glu Asp Leu LysVal Gln His Ser Ser 65 70 75 80 tac aga cag agg gcc cgg ctg ttg aag gaccag ctc tcc ctg gga aat 346 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp GlnLeu Ser Leu Gly Asn 85 90 95 gct gca ctt cag atc aca gat gtg aaa ttg caggat gca ggg gtg tac 394 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln AspAla Gly Val Tyr 100 105 110 cgc tgc atg atc agc tat ggt ggt gcc gac tacaag cga att act gtg 442 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr LysArg Ile Thr Val 115 120 125 aaa gtc aat gcc cca tac aac aaa atc aac caaaga att ttg gtt gtg 490 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln ArgIle Leu Val Val 130 135 140 gat cca gtc acc tct gaa cat gaa ctg aca tgtcag gct gag ggc tac 538 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys GlnAla Glu Gly Tyr 145 150 155 160 ccc aag gcc gaa gtc atc tgg aca agc agtgac cat caa gtc ctg agt 586 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser AspHis Gln Val Leu Ser 165 170 175 ggt aag acc acc acc acc aat tcc aag agagag gag aag ctt ttc aat 634 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg GluGlu Lys Leu Phe Asn 180 185 190 gtg acc agc aca ctg aga atc aac aca acaact aat gag att ttc tac 682 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr ThrAsn Glu Ile Phe Tyr 195 200 205 tgc act ttt agg aga tta gat cct gag gaaaac cat aca gct gaa ttg 730 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu AsnHis Thr Ala Glu Leu 210 215 220 gtc atc cca ggt aat att ctg aat gtg tccatt aaa ata tgt cta aca 778 Val Ile Pro Gly Asn Ile Leu Asn Val Ser IleLys Ile Cys Leu Thr 225 230 235 240 ctg tcc cct agc acc tagcatgatgtctgcctatc atagtcattc agtgattgtt 833 Leu Ser Pro Ser Thr 245 gaataaatgaatgaatgaat aacactatgt ttacaaaata tatcctaatt cctcacctcc 893 attcatccaaaccatattgt tacttaataa acattcagca gatatttatg gaataaaaaa 953 aaaaaaaaaaaaaaa 968 2 245 PRT Homo sapiens 2 Met Arg Ile Phe Ala Val Phe Ile PheMet Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val ProLys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu CysLys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala Leu Ile Val TyrTrp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln Phe Val His Gly Glu GluAsp Leu Lys Val Gln His Ser Ser 65 70 75 80 Tyr Arg Gln Arg Ala Arg LeuLeu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr AspVal Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser TyrGly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala ProTyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val ThrSer Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro LysAla Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 GlyLys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210215 220 Val Ile Pro Gly Asn Ile Leu Asn Val Ser Ile Lys Ile Cys Leu Thr225 230 235 240 Leu Ser Pro Ser Thr 245 3 1553 DNA Homo sapiens CDS(53)..(922) 3 cgaggctccg caccagccgc gcttctgtcc gcctgcaggg cattccagaa agatg agg 58 Met Arg 1 ata ttt gct gtc ttt ata ttc atg acc tac tgg cat ttgctg aac gca 106 Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu LeuAsn Ala 5 10 15 ttt act gtc acg gtt ccc aag gac cta tat gtg gta gag tatggt agc 154 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr GlySer 20 25 30 aat atg aca att gaa tgc aaa ttc cca gta gaa aaa caa tta gacctg 202 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu35 40 45 50 gct gca cta att gtc tat tgg gaa atg gag gat aag aac att attcaa 250 Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln55 60 65 ttt gtg cat gga gag gaa gac ctg aag gtt cag cat agt agc tac aga298 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg 7075 80 cag agg gcc cgg ctg ttg aag gac cag ctc tcc ctg gga aat gct gca346 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala 8590 95 ctt cag atc aca gat gtg aaa ttg cag gat gca ggg gtg tac cgc tgc394 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys 100105 110 atg atc agc tat ggt ggt gcc gac tac aag cga att act gtg aaa gtc442 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val 115120 125 130 aat gcc cca tac aac aaa atc aac caa aga att ttg gtt gtg gatcca 490 Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro135 140 145 gtc acc tct gaa cat gaa ctg aca tgt cag gct gag ggc tac cccaag 538 Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys150 155 160 gcc gaa gtc atc tgg aca agc agt gac cat caa gtc ctg agt ggtaag 586 Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys165 170 175 acc acc acc acc aat tcc aag aga gag gag aag ctt ttc aat gtgacc 634 Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr180 185 190 agc aca ctg aga atc aac aca aca act aat gag att ttc tac tgcact 682 Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr195 200 205 210 ttt agg aga tta gat cct gag gaa aac cat aca gct gaa ttggtc atc 730 Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu ValIle 215 220 225 cca gaa cta cct ctg gca cat cct cca aat gaa agg act cacttg gta 778 Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His LeuVal 230 235 240 att ctg gga gcc atc tta tta tgc ctt ggt gta gca ctg acattc atc 826 Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr PheIle 245 250 255 ttc cgt tta aga aaa ggg aga atg atg gat gtg aaa aaa tgtggc atc 874 Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys GlyIle 260 265 270 caa gat aca aac tca aag aag caa agt gat aca cat ttg gaggag acg 922 Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu GluThr 275 280 285 290 taatccagca ttggaacttc tgatcttcaa gcagggattctcaacctgtg gtttaggggt 982 tcatcggggc tgagcgtgac aagaggaagg aatgggcccgtgggatgcag gcaatgtggg 1042 acttaaaagg cccaagcact gaaaatggaa cctggcgaaagcagaggagg agaatgaaga 1102 aagatggagt caaacaggga gcctggaggg agaccttgatactttcaaat gcctgagggg 1162 ctcatcgacg cctgtgacag ggagaaagga tacttctgaacaaggagcct ccaagcaaat 1222 catccattgc tcatcctagg aagacgggtt gagaatccctaatttgaggg tcagttcctg 1282 cagaagtgcc ctttgcctcc actcaatgcc tcaatttgttttctgcatga ctgagagtct 1342 cagtgttgga acgggacagt atttatgtat gagtttttcctatttatttt gagtctgtga 1402 ggtcttcttg tcatgtgagt gtggttgtga atgatttcttttgaagatat attgtagtag 1462 atgttacaat tttgtcgcca aactaaactt gctgcttaatgatttgctca catctagtaa 1522 aacatggagt atttgtaaaa aaaaaaaaaa a 1553 4 290PRT Homo sapiens 4 Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr TrpHis Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu TyrVal Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro ValGlu Lys Gln Leu 35 40 45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met GluAsp Lys Asn Ile 50 55 60 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys ValGln His Ser Ser 65 70 75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp GlnLeu Ser Leu Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu GlnAsp Ala Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala AspTyr Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys IleAsn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His GluLeu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val IleTrp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr ThrThr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr Ser ThrLeu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205 Cys Thr PheArg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220 Val IlePro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 225 230 235 240Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr 245 250255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu275 280 285 Glu Thr 290 5 24 DNA Homo sapiens 5 cagctatggt ggtgccgactacaa 24 6 25 DNA Homo sapiens 6 aggtgctagg ggacagtgtt agaca 25 7 24 DNAHomo sapiens 7 cagctatggt ggtgccgact acaa 24 8 25 DNA Homo sapiens 8aggtgctagg ggacagtgtt agaca 25 9 24 DNA Homo sapiens 9 tcgcttgtagtcggcaccac cata 24 10 864 DNA Homo sapiens 10 atgcagatcc cacaggcgccctggccagtc gtctgggcgg tgctacaact gggctggcgg 60 ccaggatggt tcttagactccccagacagg ccctggaacc cccccacctt ctccccagcc 120 ctgctcgtgg tgaccgaaggggacaacgcc accttcacct gcagcttctc caacacatcg 180 gagagcttcg tgctaaactggtaccgcatg agccccagca accagacgga caagctggcc 240 gccttccccg aggaccgcagccagcccggc caggactgcc gcttccgtgt cacacaactg 300 cccaacgggc gtgacttccacatgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360 tacctctgtg gggccatctccctggccccc aaggcgcaga tcaaagagag cctgcgggca 420 gagctcaggg tgacagagagaagggcagaa gtgcccacag cccaccccag cccctcaccc 480 aggtcagccg gccagttccaaaccctggtg gttggtgtcg tgggcggcct gctgggcagc 540 ctggtgctgc tagtctgggtcctggccgtc atctgctccc gggccgcacg agggacaata 600 ggagccaggc gcaccggccagcccctgaag gaggacccct cagccgtgcc tgtgttctct 660 gtggactatg gggagctggatttccagtgg cgagagaaga ccccggagcc ccccgtgccc 720 tgtgtccctg agcagacggagtatgccacc attgtctttc ctagcggaat gggcacctca 780 tcccccgccc gcaggggctcagctgacggc cctcggagtg cccagccact gaggcctgag 840 gatggacact gctcttggcccctc 864 11 921 DNA Homo sapiens CDS (25)..(888) 11 cactctggtggggctgctcc aggc atg cag atc cca cag gcg ccc tgg cca 51 Met Gln Ile ProGln Ala Pro Trp Pro 1 5 gtc gtc tgg gcg gtg cta caa ctg ggc tgg cgg ccagga tgg ttc tta 99 Val Val Trp Ala Val Leu Gln Leu Gly Trp Arg Pro GlyTrp Phe Leu 10 15 20 25 gac tcc cca gac agg ccc tgg aac ccc ccc acc ttctcc cca gcc ctg 147 Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe SerPro Ala Leu 30 35 40 ctc gtg gtg acc gaa ggg gac aac gcc acc ttc acc tgcagc ttc tcc 195 Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys SerPhe Ser 45 50 55 aac aca tcg gag agc ttc gtg cta aac tgg tac cgc atg agcccc agc 243 Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser ProSer 60 65 70 aac cag acg gac aag ctg gcc gcc ttc ccc gag gac cgc agc cagccc 291 Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro75 80 85 ggc cag gac tgc cgc ttc cgt gtc aca caa ctg ccc aac ggg cgt gac339 Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp 9095 100 105 ttc cac atg agc gtg gtc agg gcc cgg cgc aat gac agc ggc acctac 387 Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr110 115 120 ctc tgt ggg gcc atc tcc ctg gcc ccc aag gcg cag atc aaa gagagc 435 Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser125 130 135 ctg cgg gca gag ctc agg gtg aca gag aga agg gca gaa gtg cccaca 483 Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr140 145 150 gcc cac ccc agc ccc tca ccc agg tca gcc ggc cag ttc caa accctg 531 Ala His Pro Ser Pro Ser Pro Arg Ser Ala Gly Gln Phe Gln Thr Leu155 160 165 gtg gtt ggt gtc gtg ggc ggc ctg ctg ggc agc ctg gtg ctg ctagtc 579 Val Val Gly Val Val Gly Gly Leu Leu Gly Ser Leu Val Leu Leu Val170 175 180 185 tgg gtc ctg gcc gtc atc tgc tcc cgg gcc gca cga ggg acaata gga 627 Trp Val Leu Ala Val Ile Cys Ser Arg Ala Ala Arg Gly Thr IleGly 190 195 200 gcc agg cgc acc ggc cag ccc ctg aag gag gac ccc tca gccgtg cct 675 Ala Arg Arg Thr Gly Gln Pro Leu Lys Glu Asp Pro Ser Ala ValPro 205 210 215 gtg ttc tct gtg gac tat ggg gag ctg gat ttc cag tgg cgagag aag 723 Val Phe Ser Val Asp Tyr Gly Glu Leu Asp Phe Gln Trp Arg GluLys 220 225 230 acc ccg gag ccc ccc gtg ccc tgt gtc cct gag cag acg gagtat gcc 771 Thr Pro Glu Pro Pro Val Pro Cys Val Pro Glu Gln Thr Glu TyrAla 235 240 245 acc att gtc ttt cct agc gga atg ggc acc tca tcc ccc gcccgc agg 819 Thr Ile Val Phe Pro Ser Gly Met Gly Thr Ser Ser Pro Ala ArgArg 250 255 260 265 ggc tca gct gac ggc cct cgg agt gcc cag cca ctg aggcct gag gat 867 Gly Ser Ala Asp Gly Pro Arg Ser Ala Gln Pro Leu Arg ProGlu Asp 270 275 280 gga cac tgc tct tgg ccc ctc tgaccggctt ccttggccaccagtgttctg cag 921 Gly His Cys Ser Trp Pro Leu 285 12 288 PRT Homosapiens 12 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val LeuGln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp ArgPro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr GluGly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu SerPhe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp LysLeu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp CysArg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met SerVal Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys GlyAla Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu ArgAla Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr AlaHis Pro Ser Pro Ser Pro 145 150 155 160 Arg Ser Ala Gly Gln Phe Gln ThrLeu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val LeuLeu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg GlyThr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp ProSer Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp PheGln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys ValPro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255 MetGly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280285 13 20 DNA Homo sapiens 13 gccgaagtca tctggacaag 20 14 20 DNA Homosapiens 14 tctcagtgtg ctggtcacat 20 15 20 DNA Homo sapiens 15 caccaccaccaattccaaga 20 16 23 DNA Homo sapiens 16 acgtgaccaa ggaagtgaaa gaa 23 1723 DNA Homo sapiens 17 tgccagctct tcaacagaaa cat 23 18 23 DNA Homosapiens 18 tggcaacgct gtcctgtggt cac 23 19 19 DNA Homo sapiens 19gggccgcaca agttttgat 19 20 22 DNA Homo sapiens 20 gcccttgtcc ttgatctgaaga 22 21 27 DNA Homo sapiens 21 cggacagttg gaccctgaga cttcaca 27 22 3593DNA Mus musculus CDS (17)..(889) 22 agatagttcc caaaac atg agg ata tttgct ggc att ata ttc aca gcc tgc 52 Met Arg Ile Phe Ala Gly Ile Ile PheThr Ala Cys 1 5 10 tgt cac ttg cta cgg gcg ttt act atc acg gct cca aaggac ttg tac 100 Cys His Leu Leu Arg Ala Phe Thr Ile Thr Ala Pro Lys AspLeu Tyr 15 20 25 gtg gtg gag tat ggc agc aac gtc acg atg gag tgc aga ttccct gta 148 Val Val Glu Tyr Gly Ser Asn Val Thr Met Glu Cys Arg Phe ProVal 30 35 40 gaa cgg gag ctg gac ctg ctt gcg tta gtg gtg tac tgg gaa aaggaa 196 Glu Arg Glu Leu Asp Leu Leu Ala Leu Val Val Tyr Trp Glu Lys Glu45 50 55 60 gat gag caa gtg att cag ttt gtg gca gga gag gag gac ctt aagcct 244 Asp Glu Gln Val Ile Gln Phe Val Ala Gly Glu Glu Asp Leu Lys Pro65 70 75 cag cac agc aac ttc agg ggg aga gcc tcg ctg cca aag gac cag ctt292 Gln His Ser Asn Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu 8085 90 ttg aag gga aat gct gcc ctt cag atc aca gac gtc aag ctg cag gac340 Leu Lys Gly Asn Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp 95100 105 gca ggc gtt tac tgc tgc ata atc agc tac ggt ggt gcg gac tac aag388 Ala Gly Val Tyr Cys Cys Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys 110115 120 cga atc acg ctg aaa gtc aat gcc cca tac cgc aaa atc aac cag aga436 Arg Ile Thr Leu Lys Val Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg 125130 135 140 att tcc gtg gat cca gcc act tct gag cat gaa cta ata tgt caggcc 484 Ile Ser Val Asp Pro Ala Thr Ser Glu His Glu Leu Ile Cys Gln Ala145 150 155 gag ggt tat cca gaa gct gag gta atc tgg aca aac agt gac caccaa 532 Glu Gly Tyr Pro Glu Ala Glu Val Ile Trp Thr Asn Ser Asp His Gln160 165 170 ccc gtg agt ggg aag aga agt gtc acc act tcc cgg aca gag gggatg 580 Pro Val Ser Gly Lys Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met175 180 185 ctt ctc aat gtg acc agc agt ctg agg gtc aac gcc aca gcg aatgat 628 Leu Leu Asn Val Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn Asp190 195 200 gtt ttc tac tgt acg ttt tgg aga tca cag cca ggg caa aac cacaca 676 Val Phe Tyr Cys Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr205 210 215 220 gcg gag ctg atc atc cca gaa ctg cct gca aca cat cct ccacag aac 724 Ala Glu Leu Ile Ile Pro Glu Leu Pro Ala Thr His Pro Pro GlnAsn 225 230 235 agg act cac tgg gtg ctt ctg gga tcc atc ctg ttg ttc ctcatt gta 772 Arg Thr His Trp Val Leu Leu Gly Ser Ile Leu Leu Phe Leu IleVal 240 245 250 gtg tcc acg gtc ctc ctc ttc ttg aga aaa caa gtg aga atgcta gat 820 Val Ser Thr Val Leu Leu Phe Leu Arg Lys Gln Val Arg Met LeuAsp 255 260 265 gtg gag aaa tgt ggc gtt gaa gat aca agc tca aaa aac cgaaat gat 868 Val Glu Lys Cys Gly Val Glu Asp Thr Ser Ser Lys Asn Arg AsnAsp 270 275 280 aca caa ttc gag gag acg taa gcagtgttga accctctgatcgtcgattgg 919 Thr Gln Phe Glu Glu Thr 285 290 cagcttgtgg tctgtgaaagaaagggccca tgggacatga gtccaaagac tcaagatgga 979 acctgaggga gagaaccaagaaagtgttgg gagaggagcc tggaacaacg gacatttttt 1039 ccagggagac actgctaagcaagttgccca tcagtcgtct tgggaaatgg attgagggtt 1099 cctggcttag cagctggtccttgcacagtg accttttcct ctgctcagtg ccgggatgag 1159 agatggagtc atgagtgttgaagaataagt gccttctatt tattttgagt ctgtgtgttc 1219 tcactttggg catgtaattatgactggtga attctgacga catgatagat cttaagatgt 1279 agtcaccaaa ctcaactgctgcttagcatc ctccgtaact actgatacaa gcagggaaca 1339 cagaggtcac ctgcttggtttgacaggctc ttgctgtctg actcaaataa tctttatttt 1399 tcagtcctca aggctcttcgatagcagttg ttctgtatca gccttatagg tgtcaggtat 1459 agcactcaac atctcatctcattacaatag caaccctcat caccatagca acagctaacc 1519 tctgttatcc tcacttcatagccaggaagc tgagcgacta agtcacttgc ccacagagta 1579 tcagctctca gatttctgttcttcagccac tgtcctttca ggatagaatt tgtcgttaag 1639 aaattaattt aaaaactgattattgagtag cattgtatat caatcacaac atgccttgtg 1699 cactgtgctg gcctctgagcataaagatgt acgccggagt accggtcgga catgtttatg 1759 tgtgttaaat actcagagaaatgttcatta acaaggagct tgcattttag agacactgga 1819 aagtaactcc agttcattgtctagcattac atttacctca tttgctatcc ttgccataca 1879 gtctcttgtt ctccatgaagtgtcatgaat cttgttgaat agttctttta ttttttaaat 1939 gtttctattt aaatgatattgacatctgag gcgatagctc agttggtaaa accctttcct 1999 cacaagtgtg aaaccctgagtcttatccct agaacccaca taaaaaacag ttgcgtatgt 2059 ttgtgcatgc ttttgatcccagcactaggg aggcagaggc aggcagatcc tgagctctca 2119 ttgaccaccc agcctagcctacatggttag ctccaggcct acaggagctg gcagagcctg 2179 aaaaacgatg cctagacacacacacacaca cacacacaca cacacacaca cacacacacc 2239 atgtactcat agacctaagtgcaccctcct acacatgcac acacatacaa ttcaaacaca 2299 aatcaacagg gaattgtctcagaatggtcc ccaagacaaa gaagaagaaa aacaccaaac 2359 cagctctatt ccctcagcctatcctctcta ctccttccta gaagcaacta ctattgtttt 2419 tgtatataaa tttacccaacgacagttaat atgtagaata tatattaaag tgtctgtcaa 2479 tatatattat ctctttctttctttcttcct ttctttcttt ctttctttct ttctttcttt 2539 ctttctttct ttctttctttcttccttcct tccttccttc cttccttcct tccttccttt 2599 ctttctttct ttctttttttctgtctatct gtacctaaat ggttgctcac tatgcatttt 2659 ctgtgctctt cgccctttttatttaatgta tggatattta tgctgcttcc agaatggatc 2719 taaagctctt tgtttctaggttttctcccc catccttcta ggcatctctc acactgtcta 2779 ggccagacac catgtctgctgcctgaatct gtagacacca tttataaagc acgtactcac 2839 cgagtttgta tttggcttgttctgtgtctg attaaaggga gaccatgagt ccccagggta 2899 cactgagtta ccccagtaccaagggggagc cttgtttgtg tctccatggc agaagcaggc 2959 ctggagccat tttggtttcttccttgactt ctctcaaaca cagacgcctc acttgctcat 3019 tacaggttct cctttgggaatgtcagcatt gctccttgac tgctggctgc cctggaagga 3079 gcccattagc tctgtgtgagcccttgacag ctactgcctc tccttaccac aggggcctct 3139 aagatactgt tacctagaggtcttgaggat ctgtgttctc tggggggagg aaaggaggag 3199 gaacccagaa ctttcttacagttttccttg ttctgtcaca tgtcaagact gaaggaacag 3259 gctgggctac gtagtgagatcctgtctcaa aggaaagacg agcatagccg aacccccggt 3319 ggaaccccct ctgttacctgttcacacaag cttattgatg agtctcatgt taatgtcttg 3379 tttgtatgaa gtttaagaaaatatcgggtt gggcaacaca ttctatttat tcattttatt 3439 tgaaatctta atgccatctcatggtgttgg attggtgtgg cactttattc ttttgtgttg 3499 tgtataacca taaattttattttgcatcag attgtcaatg tattgcatta atttaataaa 3559 tatttttatt tattaaaaaaaaaaaaaaaa aaaa 3593 23 290 PRT Mus musculus 23 Met Arg Ile Phe Ala GlyIle Ile Phe Thr Ala Cys Cys His Leu Leu 1 5 10 15 Arg Ala Phe Thr IleThr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Val ThrMet Glu Cys Arg Phe Pro Val Glu Arg Glu Leu 35 40 45 Asp Leu Leu Ala LeuVal Val Tyr Trp Glu Lys Glu Asp Glu Gln Val 50 55 60 Ile Gln Phe Val AlaGly Glu Glu Asp Leu Lys Pro Gln His Ser Asn 65 70 75 80 Phe Arg Gly ArgAla Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn 85 90 95 Ala Ala Leu GlnIle Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110 Cys Cys IleIle Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu 115 120 125 Lys ValAsn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp 130 135 140 ProAla Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro 145 150 155160 Glu Ala Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly 165170 175 Lys Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val180 185 190 Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe TyrCys 195 200 205 Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala GluLeu Ile 210 215 220 Ile Pro Glu Leu Pro Ala Thr His Pro Pro Gln Asn ArgThr His Trp 225 230 235 240 Val Leu Leu Gly Ser Ile Leu Leu Phe Leu IleVal Val Ser Thr Val 245 250 255 Leu Leu Phe Leu Arg Lys Gln Val Arg MetLeu Asp Val Glu Lys Cys 260 265 270 Gly Val Glu Asp Thr Ser Ser Lys AsnArg Asn Asp Thr Gln Phe Glu 275 280 285 Glu Thr 290

What is claimed is:
 1. A method for downmodulating an immune responsecomprising contacting an immune cell expressing PD-1 with an agent thatbinds to PD-1, wherein the agent comprises an antibody to PD-1 inmultivalent form, such that a negative signal is transduced via PD-1 tothereby downmodulate the immune response.
 2. The method of claim 1,wherein the agent further comprises a binding specificity for anactivating receptor on the immune cell.
 3. The method of claim 1,wherein the immune cell is selected from the group consisting of: a Tcell, a B cell, and a myeloid cell.
 4. The method of claim 2, whereinanergy is induced in the immune cell.
 5. The method of claim 2, furthercomprising contacting the immune cell with an additional agent thatdownregulates an immune response.
 6. The method of claim 1, wherein thestep of contacting occurs in vivo.
 7. The method of claim 1, wherein thestep of contacting occurs in vitro.
 8. The method of claim 1, whereinthe agent is a cross-linked antibody to PD-1.
 9. The method of claim 1,wherein the agent comprises an immobilized antibody to PD-1.